CN111954529A - Alpha polyglutamated methotrexate and uses thereof - Google Patents

Alpha polyglutamated methotrexate and uses thereof Download PDF

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CN111954529A
CN111954529A CN201980024640.7A CN201980024640A CN111954529A CN 111954529 A CN111954529 A CN 111954529A CN 201980024640 A CN201980024640 A CN 201980024640A CN 111954529 A CN111954529 A CN 111954529A
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alpha
composition
pmtx
methotrexate
cancer
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C·尼基萨
V·M·莫约
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LEAF Holdings Group LLC
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Abstract

The present disclosure relates generally to alpha-polyglutamated methotrexate, formulations containing liposomes filled with alpha-polyglutamated methotrexate, methods of making formulations containing the alpha-polyglutamated methotrexate and liposomes, and methods of treating hyperproliferative disorders (e.g., cancer) and immune system disorders (e.g., autoimmune diseases, such as rheumatoid arthritis) using formulations containing alpha-polyglutamated methotrexate and liposomes.

Description

Alpha polyglutamated methotrexate and uses thereof
Background
The present disclosure generally relates to alpha polyglutamated methotrexate compositions, including delivery vehicles, such as liposomes containing the alpha polyglutamated methotrexate compositions; as well as methods of making and using the compositions to treat diseases including hyperproliferative diseases (such as cancer), immune system disorders (including inflammatory and autoimmune diseases, such as rheumatoid arthritis), and infectious diseases (such as HIV and malaria).
Methotrexate has gained widespread clinical use worldwide as an important component of a multi-drug regimen for the treatment of Acute Lymphoblastic Leukemia (ALL), lymphoma, and solid tumors. Methotrexate (MTX) is also the disease-modifying antirheumatic drug (DMARD) most widely used as the anchor in the treatment of patients with Rheumatoid Arthritis (RA). It is used as a single agent or in combination with other DMARDs (e.g., sulfasalazine and hydroxychloroquine), and MTX use is essential in most therapeutic strategies involving biological agents (e.g., anti-TNF α and anti-CD 20 monoclonal antibodies). For the treatment of breast cancer, advanced head and neck cancer, lung and stomach cancer, osteosarcoma, non-Hodgkin lymphoma (NHL), Acute Lymphoblastic Leukemia (ALL), mycosis fungoides (cutaneous T-cell lymphoma), choriocarcinoma and chorioadenoma. Off-label cancer uses for methotrexate include the prevention of non-leukemic meningeal cancer, soft tissue sarcoma (desmoid, invasive fibromatosis), bladder cancer, Central Nervous System (CNS) lymphoma, and graft versus host disease.
MTX is also used in non-cancerous conditions such as psoriasis and rheumatoid arthritis, Inflammatory Bowel Disease (IBD), systemic inflammation, atherosclerosis, cardiovascular disease (CVD), coronary artery disease and gestational trophoblastic disease. Some off-label non-cancer uses include crohn's disease, dermatomyositis/polymyositis, ectopic pregnancy, systemic lupus erythematosus, and takayasu's arteritis.
Methotrexate (a folic acid analog, which differs from folic acid in that the hydroxyl group is replaced with an amino group at position 4 of the pteridine ring. this minor structural change creates the ability of MTX to inhibit the active catalytic site of dihydrofolate reductase (DHFR), which catalyzes the production of Tetrahydrofolate (THF) from dihydrofolate (DHF.) thus, methotrexate interferes with the synthesis of Tetrahydrofolate (THF), which serves as the primary carbon carrier for the enzymatic processes involved in the de novo synthesis of thymidylate, purine nucleotides, and the amino acids serine and methionine.
Folate is an essential cofactor that mediates the transfer of one-carbon units involved in nucleotide biosynthesis and DNA repair, the re-methylation of homocysteine (Hcy), and the methylation of DNA, proteins, and lipids. The only circulating form of folate in the blood is monoglutamate, and folate monoglutamate is the only form of folate transported across the cell membrane-likewise, the monoglutamate forms of polyglutamated antifolates (e.g., methotrexate) are transported across cell membranes. Once taken up into the cell, intracellular folate is converted to polyglutamate by the enzyme folyl poly-gamma-glutamate synthetase (FPGS).
Methotrexate is transported into cells via the Reducing Folate Carrier (RFC) system and the Folate Receptors (FR) α and β and the proton-coupled folate transporter (PCFT), which is generally the most active in a lower pH environment. RFC is a major transporter for methotrexate at physiological pH and is ubiquitously expressed in both normal and diseased cells. Thus, methotrexate treatment often suffers from dose-limiting toxicity, which is a major obstacle to cancer chemotherapy. Once inside the cell, methotrexate is polyglutamated by FPGS, which can add up to 6L-glutamyl groups in the L- γ carboxyl linkage of methotrexate. There are at least two main therapeutic objectives for L-gamma polyglutamation of methotrexate by FPGS: (1) it greatly enhances the affinity and inhibitory activity of methotrexate on DHFR; and (2) it promotes the accumulation of polyglutamated methotrexate, which, unlike methotrexate (monoglutamate), is not readily transported out of the cell by extracellular pumps.
Although targeting folate metabolism and nucleotide biosynthesis is a well established cancer therapeutic strategy, clinical efficacy is limited for MTX due to lack of tumor selectivity and the presence of primary (de novo) and acquired resistance. Like other antifolates, methotrexate plays a role in DNA and RNA synthesis and therefore has a more toxic effect on rapidly dividing cells (e.g., malignant and myeloid cells). Myelosuppression is generally a dose-limiting toxicity of methotrexate therapy and limits the clinical use of methotrexate.
Resistance to methotrexate therapy is often associated with one or more of the following: (a) increased extracellular pump activity, (b) decreased transport of MTX into cells, (c) increased DHFR activity, (d) decreased activity of folyl poly-gamma-glutamate synthetase (FPGS), and (e) increased activity of gamma-glutamyl hydrolase (GGH) which cleaves the gamma polyglutamate chain linked to folic acid and an antifolate.
The challenge of long-term (> 30 years) observations, i.e., higher levels of polyglutamate of various antifolates having greater potency than lower levels of glutamate, has been that the scientific community relies on intracellular FPGS-mediated mechanisms to convert lower levels of glutamate into their higher level forms. The present invention provides a means to deliver higher levels of antifolates in the form of polyglutamates directly into cells without having to rely on cellular mechanisms to accomplish this.
The provided alpha polyglutamated methotrexate compositions provide strategies for overcoming the pharmacological challenges associated with dose-limiting toxicity and therapeutic resistance associated with methotrexate therapy. The provided methods deliver a novel alpha polyglutamated form of methotrexate to cancer cells while (1) minimizing/reducing exposure to normal tissue cells, (2) optimizing/improving the cytotoxic effects of methotrexate-based agents on cancer cells, and (3) minimizing/reducing the impact of efflux pumps and other resistance mechanisms that limit the therapeutic efficacy of methotrexate.
Disclosure of Invention
The present disclosure relates generally to novel alpha polyglutamated Methotrexate (MTX) compositions, as well as methods of making and using the compositions to treat diseases, including hyperproliferative diseases (such as cancer), immune system disorders (such as rheumatoid arthritis), and infectious diseases (such as HIV and malaria).
In some embodiments, the present disclosure provides:
[1] a composition comprising alpha polyglutamated methotrexate, wherein at least one glutamyl group has an alpha carboxyl linkage;
[2] the composition of [1], wherein the alpha polyglutamated methotrexate comprises 1-10 glutamyl groups having an alpha carboxy linkage;
[3] the composition of [1] or [2], wherein the alpha-polyglutamated methotrexate contains 4, 5, 2-10, 4-6, or more than 5 glutamyl groups;
[4] the composition of any of [1] to [3], comprising alpha tetraglutamated methotrexate;
[5] the composition of any one of [1] to [3], comprising alpha-pentaglutamated methotrexate;
[6] the composition of any one of [1] to [3], comprising alpha-hexaglutaminated methotrexate;
[7] The composition according to any one of [1] to [6], wherein
(a) Two or more glutamyl groups have an alpha carboxyl linkage,
(b) each glutamyl group, other than the glutamyl group of methotrexate, has an alpha carboxyl linkage; or
(c) Two or more glutamyl groups have a gamma carboxyl linkage,
[8] the composition of any one of [1] to [7], wherein at least one glutamyl group has both an alpha and a gamma carboxyl linkage;
[9] the composition according to any one of [1] to [8], wherein:
(a) at least 2 glutamyl groups of the alpha polyglutamated methotrexate are in the L form,
(b) each glutamyl group of the alpha polyglutamated methotrexate is in the L form,
(c) at least 1 glutamyl group of said alpha polyglutamated methotrexate is in the D form,
(d) each glutamyl group of said alpha polyglutamated methotrexate is in the D form, in addition to the glutamyl group of methotrexate, or
(e) At least 2 glutamyl groups of the alpha polyglutamated methotrexate are in the L form, and at least 1 glutamyl group is in the D form;
[10] the composition of any one of [1] to [9], wherein the polyglutamate is linear;
[11] the composition of any one of [1] to [9], wherein the polyglutamate is branched;
[12] A liposome composition comprising alpha polyglutamated methotrexate (Lp-alpha PMTX) according to any one of [1] to [11 ];
[13] the L α PP composition of [12], wherein the α polyglutamated methotrexate comprises glutamyl in L form having an α carboxyl linkage;
[14] the Lp- α PMTX composition according to [12] or [13], wherein each glutamyl group of the α polyglutamated methotrexate is in the L form;
[15] the Lp- α PMTX composition of [12] or [13], wherein at least one glutamyl group of the α polyglutamated methotrexate is in the D form;
[16] the Lp-alpha PMTX composition of any of [12] - [15], wherein the liposome comprises alpha polyglutamated methotrexate containing 4, 5, 2-10, 4-6, or more than 5 glutamyl groups;
[17] the Lp- α PMTX composition of any one of [12] - [16], wherein at least one glutamyl group of the α polyglutamated methotrexate has a γ carboxyl linkage;
[18] the composition of any one of [12] to [17], wherein at least one glutamyl group has both an alpha and a gamma carboxyl linkage;
[19] The composition of any one of [12] to [18], the composition containing 2, 3, 4, 5, 2-10, 4-6, or more than 5 glutamyl groups having both alpha and gamma carboxyl linkages;
[20] the Lp-alpha PMTX composition of any of [12] - [19], wherein the liposome comprises alpha polyglutamated methotrexate containing alpha tetraglutamated methotrexate, alpha pentaglutamated methotrexate, or alpha hexaglutamated methotrexate;
[21] the Lp-alpha PMTX composition of any of [12] - [19], wherein the liposome comprises alpha polyglutamated methotrexate containing alpha tetraglutamated methotrexate, alpha pentaglutamated methotrexate, or alpha hexaglutamated methotrexate;
[22] the Lp-alpha PMTX composition of any of [12] - [21], wherein the polyglutamate is linear or branched;
[23] the Lp- α PMTX composition of any one of [12] - [22], wherein the liposome is pegylated (pa Lp- α PMTX);
[24] the Lp-alpha PMTX composition of any one of [12] - [23], wherein the liposomes comprise at least 1% weight/weight (w/w) of the alpha polyglutamated methotrexate, or wherein at least 1% of the starting material of alpha polyglutamated MTX is encapsulated (embedded) in the alpha PMTX during the process of preparing the Lp-alpha PMTX;
[25] The Lp- α PMTX composition of any one of [12] - [24], wherein the liposome has a diameter in the range of 20nm to 500nm or 20nm to 200 nm;
[26] the Lp- α PMTX composition of any one of [12] - [25], wherein the liposome has a diameter in the range of 80nm to 120 nm;
[27] the Lp-alpha PMTX composition of any one of [12] - [26], wherein the liposome is formed from liposome components;
[28] the Lp-alpha PMTX composition of [27], wherein the liposomal composition comprises at least one of anionic lipids and neutral lipids;
[29] the Lp- α PMTX composition according to [27] or [28], wherein the liposome component comprises at least one selected from the group consisting of: DSPE; DSPE-PEG; DSPE-PEG-maleimide; HSPC; HSPC-PEG; cholesterol; cholesterol-PEG; and cholesterol-maleimide.
[30] The Lp-alpha PMTX composition of any one of [27] - [29], wherein the liposome component comprises at least one selected from the group consisting of: DSPE; DSPE-PEG; DSPE-PEG-FITC; DSPE-PEG-maleimide; cholesterol; and HSPC;
[31] the Lp- α PMTX composition of any one of [27] to [30], wherein one or more liposomal components further comprises a steric stabilizer;
[32] The Lp- α PMTX composition of [31], wherein the steric stabilizer is at least one selected from the group consisting of: polyethylene glycol (PEG); poly-L-lysine (PLL); monosialoganglioside (GM 1); poly (vinyl pyrrolidone) (PVP); poly (acrylamide) (PAA); poly (2-methyl-2-oxazoline); poly (2-ethyl-2-oxazoline); a phosphatidylpolyglycerol; poly [ N- (2-hydroxypropyl) methacrylamide ]; amphiphilic poly-N-vinylpyrrolidone; an L-amino acid-based polymer; oligomerization of glycerol; copolymers comprising polyethylene glycol and polypropylene oxide; poloxamer 188; and polyvinyl alcohol;
[33] the Lp- α PMTX composition of [32], wherein the steric stabilizer is PEG, and the PEG has a number average molecular weight (Mn) of 200 to 5000 daltons;
[34] the Lp-alpha PMTX composition of any one of [12] to [33], wherein the liposome is anionic or neutral;
[35] the Lp-alpha PMTX composition of any of [12] - [33], wherein the liposome has a zeta potential less than or equal to zero;
[36] the Lp-alpha PMTX composition of any of [12] - [33], wherein the liposome has a zeta potential between 0 to-150 mV;
[37] The Lp-alpha PMTX composition of any of [12] - [33], wherein the liposome has a zeta potential between-30 to-50 mV;
[38] the Lp- α PMTX composition of any one of [12] to [33], wherein the liposome is cationic;
[39] the Lp-alpha PMTX composition of any one of [12] - [38], wherein the liposome has an interior space comprising the alpha polyglutamated methotrexate and a pharmaceutically acceptable aqueous carrier;
[40] the Lp- α PMTX composition of [39], wherein the pharmaceutically acceptable carrier comprises a tonicity agent such as dextrose, mannitol, glycerol, potassium chloride, sodium chloride at a concentration of greater than 1%;
[41] the Lp- α PMTX composition of [39], wherein the pharmaceutically acceptable aqueous carrier is trehalose;
[42] the Lp- α PMTX composition of [41], wherein the pharmaceutically acceptable carrier comprises 5 to 20% trehalose by weight;
[43] the Lp-alpha PMTX composition of any one of [39] - [42], wherein the pharmaceutically acceptable carrier comprises from 1% to 15% by weight of dextrose;
[44] the Lp- α PMTX composition of any of [39] - [43], wherein the interior space of the liposome comprises 5% dextrose suspended in HEPES buffer solution;
[45] The Lp-alpha PMTX composition of any one of [39] - [44], wherein the pharmaceutically acceptable carrier comprises a buffer, such as HEPES Buffered Saline (HBS) or the like, at a concentration of between 1 and 200mM and a pH of between 2 and 8;
[46] the Lp-alpha PMTX composition of any one of [39] - [45], wherein the pharmaceutically acceptable carrier comprises sodium acetate and calcium acetate in a total concentration between 50mM to 500 mM;
[47] the Lp- α PMTX composition of any of [12] - [46], wherein the interior space of the liposome has a pH of 5-8 or a pH of 6-7, or any range therebetween;
[48] the Lp-alpha PMTX composition of any one of [12] - [47], wherein the liposome comprises less than 500,000 or less than 200,000 of the alpha polyglutamated methotrexate molecules;
[49] the Lp-alpha PMTX composition of any of [12] - [48], wherein the liposome comprises between 10 to 100,000 of the alpha polyglutamated methotrexate molecules, or any range therebetween;
[50] the Lp- α PMTX composition of any one of [12] - [49], further comprising a targeting moiety, and wherein the targeting moiety has specific affinity for a surface antigen on a target cell of interest;
[51] The Lp-a PMTX composition of [50], wherein the targeting moiety is attached to one or both of PEG and the exterior of the liposome, optionally wherein targeting moiety is attached to one or both of the PEG and the exterior of the liposome by a covalent bond;
[52] the Lp- α PMTX composition of [50] or [51], wherein the targeting moiety is a polypeptide;
[53] the Lp- α PMTX composition of any one of [50] - [52], wherein the targeting moiety is an antibody or an antigen-binding fragment of an antibody;
[54]according to [50]-[53]The Lp-alpha PMTX composition of any one of, wherein, if used
Figure BDA0002715283060000041
The targeting moiety is measured by analysis to be at 0.5x 10-10To 10x 10-6An equilibrium dissociation constant (Kd) in a range binds to the surface antigen;
[55] the Lp-alpha PMTX composition of any one of [50] - [55], wherein the targeting moiety specifically binds to one or more folate receptors selected from the group consisting of: folate receptor alpha (FR-alpha), folate receptor beta (FR-beta), and folate receptor (FR-);
[56] the Lp-alpha PMTX composition of any one of [50] - [56], wherein the targeting moiety comprises one or more selected from the group consisting of: antibodies, humanized antibodies, antigen-binding fragments of antibodies, single chain antibodies, single domain antibodies, bispecific antibodies, synthetic antibodies, pegylated antibodies, and multimeric antibodies;
[57] The Lp- α PMTX composition of any one of [50] - [56], wherein each pegylated liposome comprises 1 to 1000 or 30 to 200 targeting moieties;
[58] the Lp-alpha PMTX composition of any one of [39] - [57], further comprising one or more of an immunostimulatory agent, a detectable label, and a maleimide, wherein the immunostimulatory agent, the detectable label, or the maleimide is attached to the exterior of the PEG or the liposome;
[59] the Lp- α PMTX composition of [58], wherein the immunostimulatory agent is at least one selected from the group consisting of: a protein immunostimulant; a nucleic acid immunostimulant; a chemical immunostimulant; a hapten; and an adjuvant;
[60]such as [58]]Or [59]]The Lp-alpha PMTX composition, wherein the immunostimulatory agent is at least one selected from the group consisting of: fluorescein; fluorescein Isothiocyanate (FITC); DNP; beta glucan; beta-1, 3-glucan; beta-1, 6-glucan; resolvins (e.g., resolvins D such as D)n-6DPAOr Dn-3DPAResolvin E, or T series resolvin); and Toll-like receptor (TLR) modulators, such as oxidized low density lipoproteins (e.g., OX) PAC, PGPC) and eritoran lipids (e.g., E5564);
[61] the Lp- α PMTX composition of any one of [58] to [60], wherein the immunostimulatory agent and the detectable label are the same;
[62] the Lp-alpha PMTX composition of any one of [58] to [61], further comprising a hapten;
[63] the Lp- α PMTX composition of [62], wherein the hapten comprises one or more of fluorescein or β 1, 6-glucan;
[64] the Lp-alpha PMTX composition of any one of [12] - [63], further comprising at least one cryoprotectant selected from the group consisting of: mannitol; trehalose; sorbitol; and sucrose;
[65] a targeting composition comprising the composition according to any one of [1] to [64 ];
[66] a non-targeted composition comprising the composition according to any one of [1] to [49 ];
[67] the Lp-alpha PMTX composition of any one of [12] - [66], further comprising carboplatin and/or pembrolizumab (pembrolizumab)
[68] A pharmaceutical composition comprising a liposomal alpha-polyglutamated methotrexate composition according to any one of [12] to [67 ];
[69] A pharmaceutical composition comprising an alpha polyglutamated methotrexate composition according to any one of [1] to [7 ];
[70] a composition as described in any one of [1] to [69], for use in the treatment of a disease;
[71] use of a composition as defined in any one of [1] to [70] in the manufacture of a medicament for the treatment of a disease;
[72] a method for treating or preventing a disease in a subject in need of such treatment or prevention, the method comprising administering to the subject a composition as described in any one of [1] to [70 ];
[73] a method for treating or preventing a disease in a subject in need of such treatment or prevention, the method comprising administering to the subject a liposomal α polyglutamated methotrexate composition of any one of [12] to [69 ];
[74] a method of killing a hyperproliferative cell, said method comprising contacting a hyperproliferative cell with a composition as described in any of [1] to [69 ];
[75] a method of killing hyperproliferative cells, comprising contacting hyperproliferative cells with a liposomal α polyglutamated methotrexate composition of any one of [12] to [69 ];
[76] The method of [74] or [75], wherein the hyperproliferative cell is a cancer cell, a mammalian cell, and/or a human cell;
[77] a method for treating cancer, the method comprising administering to a subject having or at risk of having cancer an effective amount of the composition of any one of [1] to [69 ];
[78] a method for treating cancer, the method comprising administering to a subject having or at risk of having cancer an effective amount of the liposomal α polyglutamated methotrexate composition of any one of [12] to [68 ];
[79] the method of [77] or [78], wherein the cancer is selected from the group consisting of: non-hematologic malignancies including, for example, lung cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer, bile duct cancer, gall bladder cancer, sarcomas (e.g., osteosarcoma), brain cancer, central nervous system cancer, and melanoma; and hematological malignancies such as leukemia, lymphoma and other B cell malignancies, myeloma and other plasma cell dyscrasias;
[80] the method of [77] or [78], wherein the cancer is a member selected from the group consisting of: breast cancer, advanced head and neck cancer, lung cancer, gastric cancer, osteosarcoma, non-hodgkin's lymphoma (NHL), Acute Lymphoblastic Leukemia (ALL), mycosis fungoides (cutaneous T-cell lymphoma), choriocarcinoma, chorioadenoma, non-leukemic meningeal cancer, soft tissue sarcoma (desmoid, invasive fibromatosis), bladder cancer, and Central Nervous System (CNS) lymphoma;
[81] The method of [77] or [78], wherein the cancer is a member selected from the group consisting of: colorectal, lung, breast, head and neck, and pancreatic cancers;
[82] the method of [77] or [78], wherein the cancer is a sarcoma, such as osteosarcoma;
[83] a method for treating cancer, the method comprising administering to a subject having or at risk of having cancer cells expressing on the surface a folate receptor bound by a targeting moiety an effective amount of the Lp- α PMTX composition of any one of [50] - [66 ].
[84] A maintenance therapy for a subject undergoing or having undergone cancer therapy, the maintenance therapy comprising administering to the subject undergoing or having undergone cancer therapy an effective amount of the composition of any one of [1] - [69 ];
[85] a maintenance therapy for a subject undergoing or having undergone cancer therapy, the maintenance therapy comprising administering to the subject undergoing or having undergone cancer therapy an effective amount of the liposomal α polyglutamated methotrexate composition of any one of [12] - [69 ];
[86] A method for treating an immune system disorder, the method comprising administering to a subject having or at risk of having an immune system disorder an effective amount of the composition of any one of [1] - [69], optionally wherein the immune system disorder is selected from: inflammation (e.g., acute and chronic inflammation), systemic inflammation, rheumatoid arthritis, Inflammatory Bowel Disease (IBD), crohn's disease, dermatomyositis/polymyositis, systemic lupus erythematosus and takayasu's arteritis, and psoriasis;
[87] a method for treating an immune system disorder, the method comprising administering to a subject having or at risk of having an immune system disorder an effective amount of the liposomal α -polyglutamated methotrexate composition of any one of [8] - [69], optionally, wherein the immune system disorder is selected from: inflammation (e.g., acute and chronic inflammation), systemic inflammation, rheumatoid arthritis, Inflammatory Bowel Disease (IBD), crohn's disease, dermatomyositis/polymyositis, systemic lupus erythematosus and takayasu's arteritis, and psoriasis;
[88] a method for treating the following diseases:
(a) an infectious disease, the method comprising administering to a subject having or at risk of having an infectious disease an effective amount of the composition according to any one of [1] to [69 ];
(b) An infectious disease, a cardiovascular disease, a metabolic disease, or another disease, the method comprising administering to a subject having or at risk of having an infectious disease, a cardiovascular disease, or another disease an effective amount of a composition according to any one of [1] - [69], wherein the disease is a member selected from: atherosclerosis, cardiovascular disease (CVD), coronary artery disease, myocardial infarction, stroke, metabolic syndrome, gestational trophoblastic disease, and ectopic pregnancy;
(c) an autoimmune disease, the method comprising administering to a subject having or at risk of having an autoimmune disease an effective amount of a composition according to any one of [1] to [69 ];
(d) rheumatoid arthritis, the method comprising administering to a subject having or at risk of having rheumatoid arthritis an effective amount of the composition according to any one of [1] to [69 ];
(e) an inflammatory disorder, the method comprising administering to a subject having or at risk of having inflammation an effective amount of a composition according to any one of [1] - [69], optionally wherein the inflammation is acute, chronic and/or systemic inflammation; or
(f) A skin condition, the method comprising administering to a subject having or at risk of having a skin condition an effective amount of the composition according to any one of [1] - [69], optionally wherein the skin condition is psoriasis;
[89] a method for treating an infectious disease, the method comprising administering to a subject having or at risk of having an infectious disease an effective amount of the liposomal α polyglutamated methotrexate composition of any one of [12] to [69 ];
[90] a method of delivering alpha polyglutamated methotrexate to a tumor that expresses a folate receptor on the surface, the method comprising: administering to a subject having the tumor an amount of an Lp-alpha PMTX composition of any one of [1] - [69] to deliver a therapeutically effective dose of alpha polyglutamated methotrexate to the tumor;
[91] a method of preparing an alpha polyglutamated methotrexate composition comprising a liposomal alpha polyglutamated methotrexate composition of any one of [12] to [69], the method comprising: forming a mixture comprising a liposome component and an alpha polyglutamated antifolate agent in solution; homogenizing the mixture to form liposomes in the solution; and processing the mixture to form liposomes containing alpha-polyglutamated methotrexate;
[92] A method of making the composition of any one of [12] to [69], the method comprising the steps of: forming a mixture comprising a liposome component and alpha-polyglutamated methotrexate in a solution; homogenizing the mixture to form liposomes in the solution; processing the mixture to form liposomes encapsulating and/or encapsulating alpha polyglutamated methotrexate; and providing a targeting moiety on the surface of the liposome, the targeting moiety having specific affinity for at least one of folate receptor alpha (FR-a), folate receptor beta (FR- β), and folate receptor (FR-);
[93] the method of [92], wherein the processing step comprises one or more of: film hydration, extrusion, online mixing, an ethanol injection technology, a freeze thawing technology, reversed phase evaporation, dynamic high-pressure micro-jet, micro-jet mixing, a multiple emulsion method, a freeze drying multiple emulsion method, 3D printing, a membrane contactor method and stirring; and/or
[94] The method of [92], wherein the processing step comprises one or more steps of altering the size of the liposomes by one or more of extrusion, high pressure microfluidization, and/or sonication steps.
In some embodiments, the present disclosure provides an alpha polyglutamated methotrexate (alpha PMTX) composition, wherein at least one glutamyl residue of the alpha polyglutamated methotrexate is linked through its alpha carboxyl group. In some embodiments, the alpha PMTX contains 2-20, 2-15, 2-10, 2-5, or more than 5 glutamyl groups (including glutamyl groups in methotrexate). In some implementations, the alpha PMTX includes two or more glutamyl groups in the form of L. In other embodiments, the alpha PMTX comprises glutamyl in the form of D. In other embodiments, the alpha PMTX comprises a glutamyl group in the D form and two or more glutamyl groups in the L form. In further embodiments, the alpha PMTX comprises two or more glutamyl groups having a gamma linkage. In some embodiments, at least one glutamyl group has both an alpha linkage and a gamma linkage.
In one embodiment, the alpha PMTX composition contains a chain of 3 glutamyl groups linked to a glutamyl group of methotrexate (i.e., tetraglutamated methotrexate). In some embodiments, the tetraglutamated MTX comprises two or more glutamyl groups in the L form. In other embodiments, the tetraglutamated MTX comprises glutamyl in the D form. In other embodiments, the tetraglutamated MTX comprises a glutamyl group in the D form and two or more glutamyl groups in the L form. In further embodiments, the tetraglutamated MTX comprises two or more glutamyl groups having a gamma linkage.
In one embodiment, the alpha PMTX composition contains a chain of 4 glutamyl groups linked to a glutamyl group of methotrexate (i.e., methotrexate pentaglutaminate). In some embodiments, the pentaglutamated MTX comprises two or more glutamyl groups in the L form. In other embodiments, the pentaglutamated MTX comprises glutamyl in the D form. In other embodiments, the pentaglutamated MTX comprises a glutamyl group in the D form and two or more glutamyl groups in the L form. In further embodiments, the pentaglutamated MTX comprises two or more glutamyl groups having a gamma linkage.
In one embodiment, the alpha PMTX composition contains a chain of 5 glutamyl groups linked to a glutamyl group of methotrexate (i.e., methotrexate hexaglutaminate). In some embodiments, the hexaglutamated MTX comprises two or more glutamyl groups in the L form. In other embodiments, the hexaglutamated MTX comprises glutamyl in the D form. In other embodiments, the hexaglutamated MTX comprises a glutamyl group in the D form and two or more glutamyl groups in the L form. In further embodiments, the hexaglutamated MTX comprises two or more glutamyl groups having a gamma linkage.
In further embodiments, the present disclosure provides compositions containing a delivery vehicle such as liposomes filled with (i.e., encapsulating) and/or otherwise associated with alpha-polyglutamated methotrexate; as well as methods of making the alpha PMTX-populating/associating delivery vehicle compositions and methods of using the alpha PMTX-populating/associating delivery vehicle compositions to deliver alpha polyglutamated methotrexate to diseased (e.g., cancerous) cells and/or targeted cells. These compositions have utility, including but not limited to, the treatment of diseases including, for example, hyperproliferative diseases (such as cancer), immune system disorders (such as rheumatoid arthritis), and infectious diseases (such as HIV and malaria). The alpha PMTX-filled/associated delivery vehicle composition provides an improvement in the efficacy and safety of delivery of methotrexate to cancer cells by providing a more cytotoxic payload (e.g., polyglutamated methotrexate) compared to the cytotoxicity of Methotrexate (MTX) administered in its monoglutamate state.
In additional embodiments, the present disclosure provides a composition comprising liposomes (Lp- α PMTX) encapsulating (filled with) α polyglutamated methotrexate. In some embodiments, the alpha polyglutamated methotrexate in Lp-alpha PMTX contains 2-20, 2-15, 2-10, 2-5, or more than 20 glutamyl groups (including glutamyl groups in methotrexate). In some embodiments, the alpha polyglutamated methotrexate in the Lp-alpha PMTX comprises two or more glutamyl groups in the L form. In other embodiments, the α polyglutamated methotrexate in Lp- α PMTX comprises a glutamyl group in the D form. In other embodiments, the α polyglutamated methotrexate in the Lp- α PMTX comprises a glutamyl group in the D form and two or more glutamyl groups in the L form. In further embodiments, the α polyglutamated methotrexate in the Lp- α PMTX comprises two or more glutamyl groups with γ linkages. In further embodiments, the alpha polyglutamated methotrexate in the Lp-alpha PMTX comprises one or more glutamyl groups having both alpha and gamma linkages. In some embodiments, the alpha polyglutamated methotrexate in the Lp-alpha PMTX comprises 2-10 glutamyl groups having both alpha and gamma linkages, or any range therebetween. In some embodiments, the polyglutamate chain of the alpha polyglutamated methotrexate is linear. In some embodiments, the polyglutamate chain of the alpha polyglutamated methotrexate is branched.
In one embodiment, the Lp-alpha PMTX composition comprises an alpha polyglutamated MTX containing a chain of 3 glutamyl groups linked to a glutamyl group of methotrexate (i.e., tetraglutamated methotrexate). In some embodiments, the tetraglutamated MTX comprises two or more glutamyl groups in the L form. In other embodiments, the tetraglutamated MTX comprises glutamyl in the D form. In other embodiments, the tetraglutamated MTX comprises a glutamyl group in the D form and two or more glutamyl groups in the L form. In further embodiments, the tetraglutamated MTX comprises two or more glutamyl groups having a gamma linkage. In some embodiments, the polyglutamate chain of the alpha polyglutamated methotrexate is linear. In some embodiments, the polyglutamate chain of the alpha polyglutamated methotrexate is branched.
In one embodiment, the Lp-alpha PMTX composition comprises an alpha polyglutamated MTX containing a chain of 4 glutamyl groups linked to the glutamyl groups of methotrexate (i.e., pentaglutamated methotrexate). In some embodiments, the pentaglutamated MTX comprises two or more glutamyl groups in the L form. In other embodiments, the pentaglutamated MTX comprises glutamyl in the D form. In other embodiments, the pentaglutamated MTX comprises a glutamyl group in the D form and two or more glutamyl groups in the L form. In further embodiments, the pentaglutamated MTX comprises two or more glutamyl groups having a gamma linkage. In some embodiments, the polyglutamate chain of the alpha polyglutamated methotrexate is linear. In some embodiments, the polyglutamate chain of the alpha polyglutamated methotrexate is branched.
In one embodiment, the Lp-alpha PMTX composition comprises an alpha polyglutamated MTX containing a chain of 5 glutamyl groups linked to a glutamyl group of methotrexate (i.e., methotrexate hexaglutaminate). In some embodiments, the hexaglutamated MTX comprises two or more glutamyl groups in the L form. In other embodiments, the hexaglutamated MTX comprises glutamyl in the D form. In other embodiments, the hexaglutamated MTX comprises a glutamyl group in the D form and two or more glutamyl groups in the L form. In further embodiments, the hexaglutamated MTX comprises two or more glutamyl groups having a gamma linkage. In some embodiments, the polyglutamate chain of the alpha polyglutamated methotrexate is linear. In some embodiments, the polyglutamate chain of the alpha polyglutamated methotrexate is branched.
In some embodiments, the Lp-alpha PMTX composition is cationic. In some embodiments, the Lp-a PMTX liposomes are cationic and have a diameter in the range of 20nm to 500nm, 20nm to 200nm, 30nm to 175nm, or 50nm to 150nm, or any range therebetween. In other embodiments, the Lp- α PMTX liposomes are cationic and the composition has a diameter in the range of 80nm to 120nm or any range therebetween. In some embodiments, the cationic Lp-alpha PMTX composition comprises at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75% (w/w) alpha polyglutamated MTX. In some embodiments, at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75% of the starting material of alpha polyglutamated MTX is encapsulated (embedded) in the cationic Lp-alpha PMTX during the process of making the Lp-alpha PMTX. In further embodiments, the α -polyglutamated methotrexate encapsulated by the liposome is in HEPES buffered solution within the liposome.
In other embodiments, the Lp- α PMTX composition is anionic or neutral. In some embodiments, the Lp-alpha PMTX composition is cationic. In some embodiments, the Lp-a PMTX liposome is anionic or neutral and has a diameter in the range of 20nm to 500nm, 20nm to 200nm, 30nm to 175nm, or 50nm to 150nm, or any range therebetween. In other embodiments, the Lp- α PMTX liposomes are anionic or neutral and the composition has a diameter in the range of 80nm to 120nm or any range therebetween. In some embodiments, the Lp-a PMTX liposome is anionic and has a diameter in the range of 20nm to 500nm, 20nm to 200nm, 30nm to 175nm, or 50nm to 150nm, or any range therebetween. In other embodiments, the Lp- α PMTX liposomes are anionic and the composition has a diameter in the range of 80nm to 120nm or any range therebetween. In some embodiments, the Lp- α PMTX liposomes are neutral and have a diameter in the range of 20nm to 500nm, 20nm to 200nm, 30nm to 175nm, or 50nm to 150nm, or any range therebetween. In other embodiments, the Lp- α PMTX liposomes are neutral and the composition has a diameter in the range of 80nm to 120nm or any range therebetween. In some embodiments, the anionic or neutral Lp- α PMTX composition comprises at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75% (w/w) of α polyglutamated MTX. In some embodiments, at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75% of the starting material of alpha polyglutamated MTX is encapsulated (embedded) in the anionic or neutral Lp-alpha PMTX during the process of making the Lp-alpha PMTX. In some embodiments, the anionic or neutral Lp- α PMTX composition comprises at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75% (w/w) α tetraglutamated MTX. In some embodiments, the anionic or neutral Lp- α PMTX composition comprises at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75% (w/w) of α pentaglutamized MTX. In some embodiments, the anionic or neutral Lp- α PMTX composition comprises at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75% (w/w) α -hexaglutamized MTX. In further embodiments, the α -polyglutamated methotrexate encapsulated by the liposome is in HEPES buffered solution within the liposome.
In further embodiments, the liposomal alpha polyglutamated methotrexate composition is pegylated (PLp-alpha PMTX).
In some embodiments, the liposomal alpha polyglutamated methotrexate composition is non-targeted (NTLp-alpha PMTX). That is, the NTLp- α PMTX compositions do not have specific affinity for an epitope expressed on the surface of the target cell of interest (e.g., an epitope on a surface antigen). In other embodiments, the non-targeted liposomal alpha polyglutamated methotrexate composition is pegylated (NTPLp-alpha PMTX).
In other embodiments, the liposomal alpha polyglutamated methotrexate composition is targeted (TLp-alpha PMTX). That is, the TLp- α PMTX composition contains a targeting moiety with specific affinity for an epitope (surface antigen) on a target cell of interest. In some embodiments, the targeting moiety of TLp- α PMTX or TPLp- α PMTX is not attached to the liposome by a covalent bond. In other embodiments, the targeting moiety of TLp-alpha PMTX or TPLp-alpha PMTX is linked to one or both of PEG and the exterior of the liposome. Targeted liposomal alpha polyglutamated methotrexate compositions (TLp-alpha PMTX and TPLp-alpha PMTX) provide further improvements in the efficacy and safety profile of methotrexate by specifically delivering alpha polyglutamated (e.g., tetraglutamated, pentaglutamated, and hexaglutamated) methotrexate to target cells such as cancer cells. In other embodiments, the targeted liposomal alpha polyglutamated methotrexate composition is pegylated (TPLp-alpha PMTX). The functions of the targeting moiety of the TLp-alpha PMTX and/or TPLp-alpha PMTX compositions include, but are not limited to, targeting liposomes to target cells of interest in vivo or in vitro; or interacts with a surface antigen for which the targeting moiety has specific affinity, and delivers the liposomal payload (α PMTX) into the cell.
Suitable targeting moieties are known in the art and include, but are not limited to, antibodies, antigen-binding antibody fragments, scaffold proteins, polypeptides and peptides. In some embodiments, the targeting moiety is a polypeptide. In other embodiments, the targeting moiety is a polypeptide comprising at least 3, 5, 10, 15, 20, 30, 40, 50, or 100 amino acid residues. In some embodiments, the targeting moiety is an antibody or antigen binding antibody fragment. In other embodiments, the targeting moiety comprises an antibody, a humanized antibody, an antigen-binding fragment of an antibody, a single chain antibody, a single domain antibody, a bispecific antibody, a synthetic antibody, a polyethyleneOne or more of a pegylated antibody and a multimeric antibody. In some embodiments, the targeting moiety has specific affinity for an epitope that is preferentially expressed on a target cell, such as a tumor cell, as compared to a normal or non-tumor cell. In some embodiments, the targeting moiety has specific affinity for an epitope on a tumor cell surface antigen that is present on a tumor cell but absent or inaccessible on a non-tumor cell. In some embodiments, e.g., using
Figure BDA0002715283060000091
The targeting moiety is measured by analysis to be at 0.5x 10-10To 10x 10-6Equilibrium dissociation constants (Kd) within the range bind to the target epitope.
In particular embodiments, the targeting moiety comprises a polypeptide that specifically binds to a folate receptor. In some embodiments, the targeting moiety is an antibody or antigen binding antibody fragment. In some embodiments, the folate receptor bound by the targeting moiety is one or more folate receptors selected from the group consisting of: folate receptor alpha (FR-alpha, FOLR1), folate receptor beta (FR-beta, FOLR2) and folate receptor (FR-, FOLR 4). In some embodiments, the folate receptor bound by the targeting moiety is folate receptor alpha (FR-alpha). In some embodiments, the folate receptor bound by the targeting moiety is folate receptor beta (FR- β). In some embodiments, the targeting moiety specifically binds FR- α and FR- β.
In further embodiments, the liposomal alpha PMTX composition comprises one or more of an immunostimulatory agent, a detectable label, and a maleimide disposed on at least one of the PEG and the exterior of the liposome. In some embodiments, the liposomal alpha PMTX composition (e.g., Lp-alpha PMTX, PLp-alpha PMTX, NTLp-alpha PMTX, NTPLp-alpha PMTX, TLp-alpha PMTX, or TPLp-alpha PMTX) is cationic. In other embodiments, the liposomal alpha PMTX composition (e.g., Lp-alpha PMTX, PLp-alpha PMTX, NTLp-alpha PMTX, NTPLp-alpha PMTX, TLp-alpha PMTX, or TPLp-alpha PMTX) is anionic or neutral. In further embodiments, the liposomes of the liposomal alpha PMTX composition (e.g., Lp-alpha PMTX, PLp-alpha PMTX, NTLp-alpha PMTX, NTPLp-alpha PMTX, TLp-alpha PMTX, or TPLp-alpha PMTX) have a diameter in the range of 20nm to 500nm, or any range therebetween. In other embodiments, the liposomes of the liposomal alpha PMTX composition have a diameter in the range of 80nm to 120nm or any range therebetween. In some embodiments, the liposomal alpha PMTX composition is pegylated (e.g., PLp-alpha PMTX, NTPLp-alpha PMTX, or TPLp-alpha PMTX). In some embodiments, the liposomal alpha PMTX composition is targeted (e.g., TLp-alpha PMTX or TPLp-alpha PMTX). In other embodiments, the liposomal alpha PMTX composition is pegylated and targeted (e.g., TPLp-alpha PMTX). In some embodiments, the liposomal α PMTX composition comprises α polyglutamated methotrexate containing 4, 5, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the liposomal α PMTX composition comprises α tetraglutamated methotrexate. In some embodiments, the liposomal α PMTX composition comprises α pentaglutamated methotrexate. In other embodiments, the liposomal α PMTX composition comprises α -hexaglutaminated methotrexate.
In some embodiments, the liposome composition comprises alpha polyglutamated methotrexate containing 4, 5, 2-10, 4-6, or more than 5 glutamyl groups and at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75% (w/w) alpha polyglutamated MTX. In some embodiments, the Lp- α PMTX composition comprises α polyglutamated methotrexate containing 4, 5, 2-10, 4-6, or more than 5 glutamyl groups and 1% -98.5% w/w α polyglutamated MTX. In some embodiments, the liposomes comprise alpha polyglutamated methotrexate containing 4, 5, 2-10, 4-6, or more than 5 glutamyl groups, and wherein at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75% of the starting material of alpha polyglutamated MTX is encapsulated (embedded) in the Lp-alpha PMTX during the process of preparing the Lp-alpha PMTX.
In some embodiments, the liposome composition comprises alpha tetraglutamated methotrexate and at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75% (w/w) alpha tetraglutamated MTX. In some embodiments, the Lp-alpha PMTX composition comprises alpha tetraglutamated methotrexate and 1% to 98.5% w/w alpha tetraglutamated MTX. In some embodiments, the liposomes comprise alpha tetraglutamated methotrexate, and wherein at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75% of the starting material of alpha tetraglutamated MTX is encapsulated (embedded) in the Lp-alpha PMTX during the process of preparing the Lp-alpha PMTX.
In some embodiments, the liposome composition comprises alpha pentaglutamated methotrexate and at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75% (w/w) alpha pentaglutamated MTX. In some embodiments, the Lp-alpha PMTX composition comprises alpha pentaglutamated methotrexate and 1% to 98.5% w/w alpha pentaglutamated MTX. In some embodiments, the liposomes comprise alpha pentaglutamated methotrexate, and wherein at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75% of the starting material of alpha pentaglutamated MTX is encapsulated (embedded) in the Lp-alpha PMTX during the process of preparing the Lp-alpha PMTX. In some embodiments, the liposome composition comprises alpha hexaglutaminated methotrexate and at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75% (w/w) alpha hexaglutamated MTX. In some embodiments, the Lp-alpha PMTX composition comprises alpha hexaglutamized methotrexate and 1% to 98.5% w/w alpha hexaglutamized MTX. In some embodiments, the liposomes comprise alpha hexaglutamated methotrexate, and wherein at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75% of the starting material of alpha pentaglutamated MTX is encapsulated (embedded) in the Lp-alpha PMTX during the process of preparing the Lp-alpha PMTX.
Also provided are liposome compositions comprising liposomes encapsulating alpha PMTX. In some embodiments, the liposome composition comprises a pegylated alpha PMTX composition. In some embodiments, the liposome composition comprises an alpha PMTX composition linked or otherwise associated with a targeting moiety. In other embodiments, the liposome composition comprises an alpha PMTX composition that is pegylated and linked or otherwise associated with a targeting moiety. In some embodiments, the liposome composition comprises alpha PMTX containing 4, 5, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the liposome composition comprises alpha-tetraglutamated methotrexate. In some embodiments, the liposome composition comprises alpha pentaglutaminated methotrexate. In other embodiments, the liposome composition comprises alpha-hexaglutaminated methotrexate.
In some embodiments, the liposome composition comprises liposomal alpha PMTX (e.g., Lp-alpha PMTX, PLp-alpha PMTX, NTLp-alpha PMTX, NTPLp-alpha PMTX, TLp-alpha PMTX, and TPLp-alpha PMTX). In some embodiments, the liposomal alpha PMTX is pegylated (e.g., NTPLp-alpha PMTX and TPLp-alpha PMTX). In some embodiments, the liposomal alpha PMTX comprises a targeting moiety (e.g., TLp-alpha PMTX or TPLp-alpha PMTX) having specific affinity for an epitope of an antigen on the surface of a target cell of interest, such as a cancer cell. In other embodiments, the liposome composition comprises pegylated liposomal alpha PMTX, and further comprises a targeting moiety (e.g., TPLp-alpha PMTX) having specific affinity for an epitope of an antigen on the surface of a target cell of interest, such as a cancer cell. In some embodiments, the liposome composition comprises a cationic liposomal alpha PMTX. In other embodiments, the liposome composition comprises liposomal alpha PMTX, which is anionic or neutral. In further embodiments, the liposome composition comprises liposomal alpha PMTX having a diameter in the range of 20nm to 500nm, 20nm to 200nm, or any range therebetween. In other embodiments, the liposomal alpha PMTX has a diameter in the range of 80nm to 120nm or any range therebetween.
Also provided are pharmaceutical compositions comprising alpha polyglutamated methotrexate (alpha PMTX), comprising a delivery vehicle, such as liposomal alpha PMTX. In some embodiments, the pharmaceutical composition comprises a pegylated alpha PMTX composition. In some embodiments, the pharmaceutical composition comprises an alpha PMTX composition linked or otherwise associated with a targeting moiety. In other embodiments, the pharmaceutical composition comprises an alpha PMTX composition that is pegylated and linked or otherwise associated with a targeting moiety. In some embodiments, the pharmaceutical composition comprises alpha PMTX containing 4, 5, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the pharmaceutical composition comprises alpha-tetraglutamated methotrexate. In some embodiments, the pharmaceutical composition comprises alpha pentaglutaminated methotrexate. In other embodiments, the pharmaceutical composition comprises alpha-hexaglutaminated methotrexate.
In some embodiments, the pharmaceutical composition comprises liposomal alpha PMTX (e.g., Lp-alpha PMTX, PLp-alpha PMTX, NTLp-alpha PMTX, NTPLp-alpha PMTX, TLp-alpha PMTX, and TPLp-alpha PMTX). In some embodiments, the liposomal alpha PMTX composition is pegylated (e.g., NTPLp-alpha PMTX and TPLp-alpha PMTX). In some embodiments, the liposomal alpha PMTX comprises a targeting moiety (e.g., TLp-alpha PMTX or TPLp-alpha PMTX) having specific affinity for an epitope of an antigen on the surface of a target cell of interest, such as a cancer cell. In other embodiments, the pharmaceutical composition comprises a pegylated liposomal alpha PMTX composition, and further comprises a targeting moiety (e.g., TPLp-alpha PMTX) having specific affinity for an epitope of an antigen on the surface of a target cell of interest, such as a cancer cell. In some embodiments, the pharmaceutical composition comprises liposomal alpha PMTX that is cationic. In other embodiments, the pharmaceutical composition comprises liposomal alpha PMTX, which is anionic or neutral. In further embodiments, the pharmaceutical composition comprises liposomal alpha PMTX having a diameter in the range of 20nm to 500nm or 20nm to 500nm, or any range therebetween. In other embodiments, the liposomal alpha PMTX composition has a diameter in the range of 80nm to 120nm or any range therebetween.
In additional embodiments, the present disclosure provides a method of modulating activation, chemokine production, or metabolic activity of a cell, comprising contacting the cell with a composition comprising an alpha polyglutamated methotrexate (alpha PMTX) composition. In some embodiments, the contacted cell is a mammalian cell. In other embodiments, the contacted cell is a human cell. In some embodiments, the contacted cell is a hyperproliferative cell. In other embodiments, the cell is an immune cell. In some embodiments, the method is performed in vivo. In other embodiments, the method is performed in vitro. In some embodiments, the alpha PMTX contains 4, 5, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the alpha PMTX composition comprises alpha tetraglutamated methotrexate. In some embodiments, the alpha PMTX composition comprises alpha pentaglutamated methotrexate. In other embodiments, the alpha PMTX composition comprises alpha hexaglutaminated methotrexate.
In additional embodiments, the present disclosure provides a method of modulating activation, chemokine production, or metabolic activity of a cell, comprising contacting the cell with a liposome comprising an alpha polyglutamated methotrexate (alpha PMTX) composition. In some embodiments, the contacted cell is a mammalian cell. In other embodiments, the contacted cell is a human cell. In some embodiments, the contacted cell is a hyperproliferative cell. In other embodiments, the cell is an immune cell. In some embodiments, the method is performed in vivo. In other embodiments, the method is performed in vitro. In some embodiments, the alpha PMTX contains 4, 5, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the alpha PMTX composition comprises alpha tetraglutamated methotrexate. In some embodiments, the alpha PMTX composition comprises alpha pentaglutamated methotrexate. In other embodiments, the alpha PMTX composition comprises alpha hexaglutaminated methotrexate.
In additional embodiments, the present disclosure provides a method of killing a cell, the method comprising contacting the cell with a composition comprising an alpha polyglutamated methotrexate (alpha PMTX) composition. In some embodiments, the contacted cell is a mammalian cell. In other embodiments, the contacted cell is a human cell. In some embodiments, the contacted cell is a hyperproliferative cell. In other embodiments, the hyperproliferative cell is a cancer cell. In other embodiments, the contacted cancer cell is a primary cell or a cell from a cell line obtained/derived from a cancer selected from the group consisting of: non-hematologic malignancies including, for example, lung cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer, bile duct cancer, gall bladder cancer, sarcomas (e.g., osteosarcoma), brain cancer, central nervous system cancer, and melanoma; and hematological malignancies such as leukemia, lymphoma and other B cell malignancies, myeloma and other plasma cell dysplasias or dyscrasias. In other embodiments, the cancer cell is a primary cell or a cell from a cell line obtained/derived from a cancer selected from: breast cancer, head and neck cancer, lung cancer, gastric cancer, osteosarcoma, non-hodgkin lymphoma (NHL), Acute Lymphoblastic Leukemia (ALL), mycosis fungoides (cutaneous T-cell lymphoma), choriocarcinoma and chorioadenoma, non-leukemic meningeal cancer, soft tissue sarcoma (desmoid, invasive fibromatosis), bladder cancer, and Central Nervous System (CNS) lymphoma. In some embodiments, the method is performed in vivo. In other embodiments, the method is performed in vitro. In some embodiments, the alpha PMTX contains 4, 5, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the alpha PMTX composition comprises alpha tetraglutamated methotrexate. In some embodiments, the alpha PMTX composition comprises alpha pentaglutamated methotrexate. In other embodiments, the alpha PMTX composition comprises alpha hexaglutaminated methotrexate.
In additional embodiments, the disclosure provides a method of killing a cell, the method comprising contacting the cell with a liposome containing alpha polyglutamated methotrexate (i.e., Lp-alpha PMTX, such as PLp-alpha PMTX, NTLp-alpha PMTX, NTPLp-alpha PMTX, TLp-alpha PMTX, or TPLp-alpha PMTX). In some embodiments, the contacted cell is a mammalian cell. In other embodiments, the contacted cell is a human cell. In some embodiments, the contacted cell is a hyperproliferative cell. In other embodiments, the hyperproliferative cell contacted is a cancer cell. In other embodiments, the cancer cell is a primary cell or a cell from a cell line obtained/derived from a cancer selected from the group consisting of: non-hematologic malignancies including, for example, lung cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer, bile duct cancer, gall bladder cancer, sarcomas (e.g., osteosarcoma), brain cancer, central nervous system cancer, and melanoma; and hematological malignancies such as leukemia, lymphoma and other B cell malignancies, myeloma and other plasma cell dysplasias or dyscrasias. In other embodiments, the cancer cell is a primary cell or a cell from a cell line obtained/derived from a cancer selected from: breast cancer, head and neck cancer, lung cancer, gastric cancer, osteosarcoma, non-hodgkin lymphoma (NHL), Acute Lymphoblastic Leukemia (ALL), mycosis fungoides (cutaneous T-cell lymphoma), choriocarcinoma and chorioadenoma, non-leukemic meningeal cancer, soft tissue sarcoma (desmoid, invasive fibromatosis), bladder cancer, and Central Nervous System (CNS) lymphoma. In some embodiments, the method is performed in vivo. In other embodiments, the method is performed in vitro. In some embodiments, the liposome contains α PMTX that contains 4, 5, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the liposome comprises alpha-tetraglutamated methotrexate. In some embodiments, the liposome comprises alpha-pentaglutaminated methotrexate. In other embodiments, the liposome comprises alpha-hexaglutaminated methotrexate.
In additional embodiments, the present disclosure provides a method for treating cancer, the method comprising administering to a subject having or at risk of having cancer an effective amount of a delivery vehicle (e.g., an immunoconjugate or liposome) comprising alpha polyglutamated methotrexate. In some embodiments, the delivery vehicle is an antibody-containing immunoconjugate (including, e.g., a full-length IgG antibody, a bispecific antibody, or an scFv). In some embodiments, the delivery vehicle is a liposome (e.g., Lp- α PMTX, such as PLp- α PMTX, NTLp- α PMTX, NTPLp- α PMTX, TLp- α PMTX, or TPLp- α PMTX). In some embodiments, the administered delivery vehicle is pegylated. In some embodiments, the administered delivery vehicle is non-pegylated. In further embodiments, the administered delivery vehicle comprises a targeting moiety having specific affinity for an epitope of an antigen on the surface of the cancer cell. In further embodiments, the delivery vehicle comprises a targeting moiety that specifically binds to a cell surface antigen selected from the group consisting of: GONMB, TACTD 2(TROP2), CEACAM5, EPCAM, folate receptor (e.g., folate receptor-alpha, folate receptor-beta, or folate receptor-), mucin 1(MUC-1), MUC-6, STEAP1, mesothelin, bindin 4, ENPP3, Guanylate Cyclase C (GCC), SLC44A4, NaPi2B, CD70(TNFSF7), CA9 (carbonic anhydrase), 5T4(TPBG), SLTRK6, SC-16, tissue factor, LIV-1(ZIP6), CGEN-15027, Pcadherin, fibronectin ectodomain B (ED-B), VEGFR2(CD309), tenascin, collagen IV, periostin, endothelin receptor, HER2, HER 72, EGFR, EGFRvIII, FGFR 72, FGFR3, CD ZD3, CD 36ZD 3, CD3, FGFR3, CD 36ZD 3, CD 36ZD 3, CD, CD19, CD20, CD22, CD26, CD27L, CD28, CD30, CD33, CD34, CD37, CD38, CD40, CD44, CD56, CD70, CD74, CD79, CD79b, CD98, CD105, CD133, CD138, cripto, IGF-1R, IGF-2R, IGF, EphA receptor, EphB receptor, EphA R, IGF, EphB R, IGF, EphA R, IGF, EphB R, IGF, EphUFB R, IGF, EphB R, IGF, integrin (for example, integrin. alpha. v. beta.3,. beta.5 or. alpha. v. beta.6), C antigen, Trk, VEGFR 72, PGR, VEGF-PGR, VEGF-R, IGF, PGRE, PGR, VEGF-R, IGF, PGR, and PG. In some embodiments, the delivery vehicle comprises a targeting moiety that specifically binds to one or more cell surface antigens (such as neoantigens) derived from or determined to be expressed on a cancer (tumor) in a particular subject. In some embodiments, the targeting moiety specifically binds to one or more cell surface antigens (such as neoantigens) derived from or determined to be expressed on a tumor in a particular subject. In some embodiments, the targeting moiety is an antibody or antigen binding antibody fragment. In some embodiments, the administered delivery vehicle comprises alpha PMTX containing 4, 5, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the administered delivery vehicle comprises α -tetraglutamated methotrexate. In some embodiments, the administered delivery vehicle comprises alpha-pentaglutaminated methotrexate. In other embodiments, the administered delivery vehicle comprises alpha-hexaglutaminated methotrexate. In some embodiments, the administered delivery vehicle comprises L α polyglutamated methotrexate. In some embodiments, the administered delivery vehicle comprises D α polyglutamated methotrexate. In other embodiments, the administered delivery vehicle comprises L and D α polyglutamated methotrexate. In some embodiments, the cancer is selected from the group consisting of: non-hematologic malignancies including, for example, lung cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer, bile duct cancer, gall bladder cancer, sarcomas (e.g., osteosarcoma), brain cancer, central nervous system cancer, and melanoma; and hematological malignancies such as leukemia, lymphoma and other B cell malignancies, myeloma and other plasma cell dysplasias or dyscrasias. In other embodiments, the cancer cell is a primary cell or a cell from a cell line obtained/derived from a cancer selected from: breast cancer, head and neck cancer, lung cancer, gastric cancer, osteosarcoma, non-hodgkin lymphoma (NHL), Acute Lymphoblastic Leukemia (ALL), mycosis fungoides (cutaneous T-cell lymphoma), choriocarcinoma and chorioadenoma, non-leukemic meningeal cancer, soft tissue sarcoma (desmoid, invasive fibromatosis), bladder cancer, and Central Nervous System (CNS) lymphoma.
In additional embodiments, the disclosure provides a method for treating cancer, the method comprising administering to a subject having or at risk of having cancer an effective amount of liposomes comprising alpha polyglutamated methotrexate (e.g., Lp-alpha PMTX, such as PLp-alpha PMTX, NTLp-alpha PMTX, NTPLp-alpha PMTX, TLp-alpha PMTX, or TPLp-alpha PMTX). In some embodiments, the liposome is pegylated. In some embodiments, the liposome is non-pegylated. In additional embodiments, the liposome comprises a targeting moiety having specific affinity for an epitope of an antigen on the surface of a cancer cell. In further embodiments, the liposome comprises a targeting moiety that specifically binds to a cell surface antigen selected from the group consisting of: GONMB, TACTD 2(TROP2), CEACAM5, EPCAM, folate receptor (e.g., folate receptor-alpha, folate receptor-beta, or folate receptor-), mucin 1(MUC-1), MUC-6, STEAP1, mesothelin, bindin 4, ENPP3, Guanylate Cyclase C (GCC), SLC44A4, NaPi2B, CD70(TNFSF7), CA9 (carbonic anhydrase), 5T4(TPBG), SLTRK6, SC-16, tissue factor, LIV-1(ZIP6), CGEN-15027, Pcadherin, fibronectin ectodomain B (ED-B), VEGFR2(CD309), tenascin, collagen IV, periostin, endothelin receptor, HER2, HER 72, EGFR, EGFRvIII, FGFR 72, FGFR3, CD ZD3, CD 36ZD 3, CD3, FGFR3, CD 36ZD 3, CD 36ZD 3, CD, CD19, CD20, CD22, CD26, CD27L, CD28, CD30, CD33, CD34, CD37, CD38, CD40, CD44, CD56, CD70, CD74, CD79, CD79b, CD98, CD105, CD133, CD138, cripto, IGF-1R, IGF-2R, IGF, EphA receptor, EphB receptor, EphA R, IGF, EphB R, IGF, EphA R, IGF, EphB R, IGF, EphUFB R, IGF, EphB R, IGF, integrin (for example, integrin. alpha. v. beta.3,. beta.5 or. alpha. v. beta.6), C antigen, Trk, VEGFR 72, PGR, VEGF-PGR, VEGF-R, IGF, PGRE, PGR, VEGF-R, IGF, PGR, and PG. Also included are uses of cancer stem cell targeting moieties, such as those targeting CD34, CD133 and CD44, CD138, and CD 15. In some embodiments, the liposome comprises a targeting moiety that specifically binds to one or more cell surface antigens (such as neoantigens) derived from or determined to be expressed on a tumor in a particular subject. In some embodiments, the targeting moiety is an antibody or antigen binding antibody fragment. In some embodiments, the liposome comprises alpha PMTX containing 4, 5, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the liposome comprises alpha-tetraglutamated methotrexate. In some embodiments, the liposome comprises alpha pentaglutaminated methotrexate. In other embodiments, the liposome comprises alpha-hexaglutaminated methotrexate. In some embodiments, the liposome comprises L α polyglutamated methotrexate. In some embodiments, the liposome comprises D α polyglutamated methotrexate. In some embodiments, the liposome comprises L and D α polyglutamated methotrexate. In some embodiments, the cancer is selected from the group consisting of: lung cancer (e.g., non-small cell lung cancer), pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, renal cancer, biliary cancer, gall bladder cancer, sarcoma (e.g., osteosarcoma), brain cancer, central nervous system cancer, melanoma, and hematologic malignancies (e.g., leukemia or lymphoma).
In additional embodiments, the present disclosure provides a method for treating cancer, the method comprising administering to a subject having or at risk of having cancer an effective amount of a liposome composition comprising liposomes comprising alpha polyglutamated methotrexate and a targeting moiety having specific affinity for an epitope of an antigen on the surface of the cancer. In some embodiments, the liposome comprises a targeting moiety that specifically binds to a cell surface antigen selected from the group consisting of: GONMB, TACTD 2(TROP2), CEACAM5, EPCAM, folate receptor (e.g., folate receptor-alpha, folate receptor-beta, or folate receptor-), mucin 1(MUC-1), MUC-6, STEAP1, mesothelin, bindin 4, ENPP3, Guanylate Cyclase C (GCC), SLC44A4, NaPi2B, CD70(TNFSF7), CA9 (carbonic anhydrase), 5T4(TPBG), SLTRK6, SC-16, tissue factor, LIV-1(ZIP6), CGEN-15027, Pcadherin, fibronectin ectodomain B (ED-B), VEGFR2(CD309), tenascin, collagen IV, periostin, endothelin receptor, HER2, HER 72, EGFR, EGFRvIII, FGFR 72, FGFR3, CD ZD3, CD 36ZD 3, CD3, FGFR3, CD 36ZD 3, CD 36ZD 3, CD, CD19, CD20, CD22, CD26, CD27L, CD28, CD30, CD33, CD34, CD37, CD38, CD40, CD44, CD56, CD70, CD74, CD79, CD79b, CD98, CD105, CD133, CD138, cripto, IGF-1R, IGF-2R, IGF, EphA receptor, EphB receptor, EphA R, IGF, EphB R, IGF, EphA R, IGF, EphB R, IGF, EphUFB R, IGF, EphB R, IGF, integrin (for example, integrin. alpha. v. beta.3,. beta.5 or. alpha. v. beta.6), C antigen, Trk, VEGFR 72, PGR, VEGF-PGR, VEGF-R, IGF, PGRE, PGR, VEGF-R, IGF, PGR, and PG. In some embodiments, the administered liposomes comprise a targeting moiety that specifically binds to one or more cell surface antigens (such as neoantigens) derived from or determined to be expressed on a tumor of a particular subject. In some embodiments, the administered liposome composition comprises pegylated liposomes (e.g., TPLp-alpha PMTX). In some embodiments, the administered liposome composition comprises non-pegylated liposomes. In some embodiments, the liposomes of the administered liposome composition comprise alpha PMTX containing 4, 5, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the liposomes of the administered liposome composition comprise alpha-tetraglutamated methotrexate. In some embodiments, the liposomes of the administered liposome composition comprise alpha-pentaglutaminated methotrexate. In other embodiments, the liposomes of the administered liposome composition comprise alpha-hexaglutaminated methotrexate. In some embodiments, the liposome composition is administered to treat a cancer selected from the group consisting of: lung cancer (e.g., non-small cell lung cancer), pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer, bile duct cancer, gall bladder cancer, sarcomas (e.g., osteosarcoma), brain cancer, central nervous system cancer, melanoma, leukemia, lymphoma and other B cell malignancies, myeloma and other plasma cell dysplasias or cachexia. In other embodiments, the cancer cell is a primary cell or a cell from a cell line obtained/derived from a cancer selected from: breast cancer, head and neck cancer, lung cancer, gastric cancer, osteosarcoma, non-hodgkin lymphoma (NHL), Acute Lymphoblastic Leukemia (ALL), mycosis fungoides (cutaneous T-cell lymphoma), choriocarcinoma and chorioadenoma, non-leukemic meningeal cancer, soft tissue sarcoma (desmoid, invasive fibromatosis), bladder cancer, and Central Nervous System (CNS) lymphoma.
In additional embodiments, the present disclosure provides a method for treating cancer, the method comprising administering to a subject having or at risk of having a cancer that expresses a folate receptor on the cell surface thereof an effective amount of a liposomal composition, wherein the liposomal composition comprises liposomes comprising (a) alpha polyglutamated methotrexate (alpha PMTX) and (b) a targeting moiety having specific binding affinity for a folate receptor. In some embodiments, the targeting moiety has specific binding affinity for folate receptor alpha (FR-alpha), folate receptor beta (FR-beta), and/or folate receptor (FR-). In some embodiments, the targeting moiety has specific binding affinity for folate receptor alpha (FR-alpha) and folate receptor beta (FR-beta). In some embodiments, the administered liposome composition comprises pegylated liposomes (e.g., TPLp-alpha PMTX). In some embodiments, the administered liposome composition comprises non-pegylated liposomes. In some embodiments, the liposomes of the administered liposome composition comprise alpha PMTX containing 4, 5, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the liposomes of the administered liposome composition comprise alpha-tetraglutamated methotrexate. In some embodiments, the liposomes of the administered liposome composition comprise alpha-pentaglutaminated methotrexate. In other embodiments, the liposomes of the administered liposome composition comprise alpha-hexaglutaminated methotrexate. In some embodiments, the liposome composition is administered to treat a cancer selected from the group consisting of: non-hematologic malignancies including, for example, lung cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer, bile duct cancer, gall bladder cancer, sarcomas (e.g., osteosarcoma), brain cancer, central nervous system cancer, and melanoma; and hematological malignancies such as leukemia, lymphoma and other B cell malignancies, myeloma and other plasma cell dysplasias or dyscrasias. In other embodiments, the cancer cell is a primary cell or a cell from a cell line obtained/derived from a cancer selected from: breast cancer, head and neck cancer, lung cancer, gastric cancer, osteosarcoma, non-hodgkin lymphoma (NHL), Acute Lymphoblastic Leukemia (ALL), mycosis fungoides (cutaneous T-cell lymphoma), choriocarcinoma and chorioadenoma, non-leukemic meningeal cancer, soft tissue sarcoma (desmoid, invasive fibromatosis), bladder cancer, and Central Nervous System (CNS) lymphoma.
In additional embodiments, the present disclosure provides a method for cancer maintenance therapy, the method comprising administering to a subject undergoing or having undergone cancer therapy an effective amount of a liposome composition comprising liposomes containing alpha polyglutamated methotrexate (Lp-alpha PMTX). In some embodiments, the liposome composition administered is PLp- α PMTX, NTLp- α PMTX, NTPLp- α PMTX, TLp- α PMTX or TPLp- α PMTX. In some embodiments, the administered liposome composition comprises pegylated liposomes (e.g., PLp- α PMTX, NTPLp- α PMTX, or TPLp- α PMTX). In some embodiments, the administered liposome composition comprises a targeted liposome (e.g., TLp-alpha PMTX or TPLp-alpha PMTX). In some embodiments, the administered liposome composition comprises pegylated and targeted liposomes (e.g., TPLp-alpha PMTX). In some embodiments, the liposomes of the administered liposome composition comprise alpha polyglutamated methotrexate containing 4, 5, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the liposomes of the administered liposome composition comprise alpha-tetraglutamated methotrexate. In some embodiments, the liposomes of the administered liposome composition comprise alpha-pentaglutaminated methotrexate. In other embodiments, the liposomes of the administered liposome composition comprise alpha-hexaglutaminated methotrexate.
In additional embodiments, the present disclosure provides a method for treating an immune system disorder, the method comprising administering to a subject having or at risk of having an immune system disorder an effective amount of a liposomal composition comprising liposomes comprising alpha polyglutamated methotrexate (e.g., Lp-alpha PMTX, PLp-alpha PMTX, NTLp-alpha PMTX, NTPLp-alpha PMTX, TLp-alpha PMTX, or TPLp-alpha PMTX). In some embodiments, the liposome composition is administered to treat an autoimmune disease. In another embodiment, the liposome composition is administered to treat rheumatoid arthritis. In some embodiments, the administered liposome composition comprises pegylated liposomes (e.g., PLp- α PMTX, NTPLp- α PMTX, or TPLp- α PMTX). In some embodiments, the administered liposome composition comprises a targeting liposome (e.g., TLp- α PMTX or TPLp- α PMTX) containing a targeting moiety with specific affinity for a surface antigen on a target cell (e.g., an immune cell) of interest. In other embodiments, the administered liposome composition comprises pegylated and targeted liposomes (e.g., TPLp-alpha PMTX)). In some embodiments, the liposomes of the administered liposome composition comprise alpha-pentaglutamated methotrexate containing 4, 5, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the liposomes of the administered liposome composition comprise alpha-tetraglutamated methotrexate. In some embodiments, the liposomes of the administered liposome composition comprise alpha-pentaglutaminated methotrexate. In other embodiments, the liposomes of the administered liposome composition comprise alpha-hexaglutaminated methotrexate. In some embodiments, the immune system disorder is selected from: inflammation (e.g., acute and chronic inflammation), systemic inflammation, rheumatoid arthritis, Inflammatory Bowel Disease (IBD), crohn's disease, dermatomyositis/polymyositis, systemic lupus erythematosus, and takayasu's arteritis, and psoriasis.
In additional embodiments, the disclosure provides a method for treating an autoimmune disease, the method comprising administering to a subject having or at risk of having an inflammatory disorder an effective amount of a liposomal composition comprising liposomes comprising alpha polyglutamated methotrexate (e.g., Lp-alpha PMTX, PLp-alpha PMTX, NTLp-alpha PMTX, NTPLp-alpha PMTX, TLp-alpha PMTX or TPLp-alpha PMTX). In some embodiments, the administered liposome composition comprises pegylated liposomes (e.g., PLp- α PMTX, NTPLp- α PMTX, or TPLp- α PMTX). In some embodiments, the administered liposome composition comprises a targeting liposome (e.g., TLp- α PMTX or TPLp- α PMTX) containing a targeting moiety with specific affinity for a surface antigen on a target cell (e.g., an immune cell) of interest. In other embodiments, the administered liposome composition comprises pegylated and targeted liposomes (e.g., TPLp-alpha PMTX)). In some embodiments, the liposomes of the administered liposome composition comprise alpha-pentaglutamated methotrexate containing 4, 5, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the liposomes of the administered liposome composition comprise alpha-tetraglutamated methotrexate. In some embodiments, the liposomes of the administered liposome composition comprise alpha-pentaglutaminated methotrexate. In other embodiments, the liposomes of the administered liposome composition comprise alpha-hexaglutaminated methotrexate. In some embodiments, the autoimmune disorder is selected from: rheumatoid arthritis, Inflammatory Bowel Disease (IBD), crohn's disease, systemic lupus erythematosus, and psoriasis.
In additional embodiments, the present disclosure provides a method for treating an inflammatory disorder, the method comprising administering to a subject having or at risk of having an inflammatory disorder an effective amount of a liposomal composition comprising liposomes comprising alpha polyglutamated methotrexate (e.g., Lp-alpha PMTX, PLp-alpha PMTX, NTLp-alpha PMTX, NTPLp-alpha PMTX, TLp-alpha PMTX or TPLp-alpha PMTX). In some embodiments, the administered liposome composition comprises pegylated liposomes (e.g., PLp- α PMTX, NTPLp- α PMTX, or TPLp- α PMTX). In some embodiments, the administered liposome composition comprises a targeting liposome (e.g., TLp- α PMTX or TPLp- α PMTX) containing a targeting moiety with specific affinity for a surface antigen on a target cell (e.g., an immune cell) of interest. In other embodiments, the administered liposome composition comprises pegylated and targeted liposomes (e.g., TPLp-alpha PMTX)). In some embodiments, the liposomes of the administered liposome composition comprise alpha-pentaglutamated methotrexate containing 4, 5, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the liposomes of the administered liposome composition comprise alpha-tetraglutamated methotrexate. In some embodiments, the liposomes of the administered liposome composition comprise alpha-pentaglutaminated methotrexate. In other embodiments, the liposomes of the administered liposome composition comprise alpha-hexaglutaminated methotrexate. In some embodiments, the inflammatory disorder is selected from the following: acute inflammation, chronic inflammation, systemic inflammation, rheumatoid arthritis, Inflammatory Bowel Disease (IBD), crohn's disease, dermatomyositis/polymyositis, and systemic lupus erythematosus.
The present disclosure also provides a method of delivering alpha-polyglutamated methotrexate to a site of inflammation in a subject, the method comprising: administering to a subject having inflammation a composition comprising alpha polyglutamated methotrexate (L-alpha PMTX) and a targeting moiety having specific binding affinity for an epitope on a surface antigen located on cells that are inflammatory or otherwise affect inflammation (e.g., via pro-inflammatory cytokine production). In some embodiments, the administered targeting moiety is associated with a delivery vehicle. In some embodiments, the delivery vehicle is an antibody or antigen-binding fragment of an antibody. In other embodiments, the delivery vehicle is a liposome. In other embodiments, the antibody, antigen-binding antibody fragment, or liposome is a pegylated liposome (e.g., TPLp- α PMTX). In some embodiments, the administered composition comprises alpha polyglutamated methotrexate containing 4, 5, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the administered composition comprises alpha-tetraglutamated methotrexate. In some embodiments, the administered composition comprises alpha pentaglutamated methotrexate. In other embodiments, the administered composition comprises alpha-hexaglutaminated methotrexate.
The present disclosure also provides a method of delivering alpha-polyglutamated methotrexate to tumor cancer cells, the method comprising: administering to a subject having the tumor a composition comprising alpha polyglutamated methotrexate (L-alpha PMTX) and a targeting moiety having specific binding affinity for an epitope on a surface antigen on a tumor cell or a cancer cell. In some embodiments, the administered targeting moiety is associated with a delivery vehicle. In some embodiments, the delivery vehicle is an antibody or antigen-binding fragment of an antibody. In other embodiments, the delivery vehicle is a liposome. In other embodiments, the antibody, antigen-binding antibody fragment, or liposome is a pegylated liposome (e.g., TPLp- α PMTX). In some embodiments, the administered composition comprises alpha polyglutamated methotrexate containing 4, 5, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the administered composition comprises alpha-tetraglutamated methotrexate. In some embodiments, the administered composition comprises alpha pentaglutamated methotrexate. In other embodiments, the administered composition comprises alpha-hexaglutaminated methotrexate.
In additional embodiments, the present disclosure provides a method of preparing a liposome composition comprising a liposomal alpha polyglutamated methotrexate (alpha PMTX) composition, the method comprising: forming a mixture comprising a liposome component and alpha-polyglutamated methotrexate in a solution; homogenizing the mixture to form liposomes in the solution; and processing the mixture to form liposomes containing polyglutamated methotrexate. In some embodiments, the alpha polyglutamated methotrexate contains 4, 5, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the polyglutamated methotrexate composition comprises α -tetraglutamated methotrexate. In some embodiments, the polyglutamated methotrexate composition comprises α -pentaglutamated methotrexate. In other embodiments, the polyglutamated methotrexate composition comprises alpha-hexaglutamated methotrexate.
In one embodiment, the present disclosure provides a kit comprising an alpha polyglutamated methotrexate composition and/or an alpha PMTX delivery vehicle, such as liposomes containing an alpha PMTX and an alpha PMTX immunoconjugate (e.g., ADC) described herein.
Drawings
Fig. 1A-1L show the chemical formulas for methotrexate (fig. 1A), exemplary alpha methotrexate alpha polyglutamate, methotrexate diglutamate (fig. 1B), methotrexate triglutamate (fig. 1C and 1D), methotrexate tetraglutamate (fig. 1E and 1F), methotrexate pentaglutamate (fig. 1G and 1H), methotrexate hexaglutamate (fig. 1I and 1J), methotrexate heptaglutamate (fig. 1K and 1L), methotrexate octaglutamate (fig. 1M and 1N), exemplary alpha methotrexate polyglutamate (fig. 1O), and exemplary methotrexate analogs (fig. 1P and 1Q). Fig. 1R-1U show a depiction of exemplary branched methotrexate polyglutamate structures, including a depiction of branched polyglutamate with a gamma glutamyl backbone and alpha glutamyl branches (fig. 1S) and branched polyglutamate with an alpha glutamyl backbone and gamma glutamyl branches (fig. 1T).
Figure 2 shows the relative potency of liposomal pemetrexed α -L hexaglutamate (liposomal aG6) and its mirror liposomal α -D hexaglutamate (liposomal aDG6) relative to pemetrexed within 48 hours after exposure to cancer cell lines SW620(CRC), HT-29 (colon cancer), H1806 (triple negative breast cancer), OAW28 (ovarian cancer), H292(NSCLC, adenocarcinoma subtype), and H2342(NSCLC, adenocarcinoma subtype).
Figure 3 shows exemplary dose response relationships of free pemetrexed L- γ -hexaglutamate (gG6), liposomal pemetrexed L- γ -hexaglutamate (liposomal gG6), pemetrexed, and folate receptor α -targeting antibody (FR1Ab) liposomal pemetrexed L- γ -hexaglutamate (liposomal gG6-FR1Ab) in NCI H2342 non-small cell lung cancer (NSCLC), adenocarcinoma subtypes, depicted as the percentage of viable cells after 48 hours of treatment. Folate receptor alpha targeting liposomes containing alpha polyglutamated pemetrexed are also expected to successfully target and reduce viability of NCI H2342 non-small cell lung cancer cells.
Figure 4 shows exemplary dose response relationships of free pemetrexed L- γ -hexaglutamate (gG6), liposomal pemetrexed L- γ -hexaglutamate (liposomal gG6), pemetrexed, and folate receptor α -targeting antibody (FR1Ab) liposomal pemetrexed L- γ -hexaglutamate (liposomal gG6-FR1Ab) in HT-29 (colon cancer) at 48 hours. Folate receptor alpha targeting liposomes containing alpha polyglutamated pemetrexed are also expected to successfully target and reduce viability of HT-29 (colon cancer) cells.
Figure 5 shows the therapeutic effect on HCC1806 triple negative breast cancer cells within 48 hours after exposure to liposomal pemetrexed α -L hexaglutamate (Lps Hexa aG6), liposomal pemetrexed α -D hexaglutamate (Lps Hexa agg 6), and pemetrexed.
Figure 6 shows the therapeutic effect on OAW28 ovarian cancer cells within 48 hours after exposure to liposomal pemetrexed α -L hexaglutamate (Lps Hexa aG6), liposomal pemetrexed α -D hexaglutamate (Lps Hexa agg 6), and pemetrexed.
Figure 7 shows the therapeutic effect on H292 non-small cell lung cancer cells within 48 hours after exposure to liposomal pemetrexed α -L hexaglutamate (Lps Hexa aG6), liposomal pemetrexed α -D hexaglutamate (Lps Hexa agg 6) compared to pemetrexed.
Figure 8 shows the therapeutic effect on H292 non-small cell lung cancer cells within 48 hours after exposure to various dose levels of liposomal pemetrexed α -L-hexaglutamate (liposomal aG6), liposomal pemetrexed α -D-hexaglutamate (liposomal aDG6), and pemetrexed in the range of 16 to 128 nM. The liposomal pemetrexed aG6 formulation outperformed pemetrexed in inhibiting H292 non-small cell lung cancer cells at each dose range tested.
Figure 9 shows the therapeutic effect on HCC1806 triple negative breast cancer cells within 48 hours after exposure to various dose levels of liposomal pemetrexed α -L-hexaglutamate (liposomal aG6), liposomal pemetrexed α -D-hexaglutamate (liposomal aDG6), and pemetrexed in the range of 16 to 128 nM. At each dose tested, the liposomal pemetrexed aG6 formulation outperformed pemetrexed in inhibiting HCC1806 triple negative breast cancer cells.
Figure 10 shows the therapeutic effect of liposomal pemetrexed α -L-hexametaphosphate (liposomal aG6), liposomal α -D hexametaphosphate (liposomal aDG6), and pemetrexed on OAW28 ovarian cancer cells within 48 hours post exposure after exposure to a range of concentrations. At 128nM, pemetrexed appeared to be more effective than the liposomal pemetrexed aG6 liposome formulation, while the liposomal formulations at 32nM and 64nM had better therapeutic effect than pemetrexed; at 16nM, liposomal pemetrexed aG6 was similar in therapeutic effect to pemetrexed.
Figure 11 shows the toxicity of liposomal pemetrexed α -L hexaglutamate (liposomal aG6), liposomal pemetrexed α -D hexaglutamate (liposomal aDG6), and pemetrexed on differentiated human neutrophils at 64nM, 128nM, and 264 nM. The figures show that liposomal pemetrexed aG6 is significantly less toxic to differentiated human neutrophils compared to pemetrexed.
Figure 12 shows the effect of liposomal pemetrexed α -L-hexaglutamate (liposomal aG6), liposomal α -D-hexaglutamate (liposomal aDG6), and pemetrexed on neutrophils (differentiated from CD34+ cells) within 48 hours after exposure to various dose levels of the respective agents in the range of 16 to 128 nM.
Figure 13 shows the effect of liposomal pemetrexed α -L hexaglutamate (liposomal aG6), liposomal pemetrexed α -D hexaglutamate (liposomal aDG6), and pemetrexed on AML12 hepatocytes within 48 hours after exposure to 16nM, 32nM, and 64nM and 128nM of the respective agents. Strikingly, at the dose levels tested, there did not appear to be any toxicity to AML12 hepatocytes after treatment with liposomal pemetrexed aG6 at any liposomal dose. In contrast, pemetrexed treatment resulted in an approximately 40% reduction in AML12 hepatocyte counts at all doses studied.
Figure 14 shows the effect of liposomal pemetrexed α -L hexaglutamate (liposomal aG6), liposomal pemetrexed α -D hexaglutamate (liposomal aDG6), and pemetrexed on CCD841 colonic epithelial cells within 48 hours after exposure to 16nM, 32nM, and 64nM and 128nM of the respective agents. At all concentrations tested, pemetrexed resulted in a reduction in the number of CCD841 colonic epithelial cells of about > 50%, compared to about 20% or less after treatment with each liposome formulation tested.
FIG. 15 depicts the structures of polyglutamate antifolate, Cisplatin (CDDP) and two potential aG 6-cisplatin complexes. The pH-dependent formation of interchain and/or intrachain coordination between polyglutamated antifolate and the carboxyl groups of cisplatin may break down into individual aG6 and cisplatin molecules upon encountering the acidic pH of lysosomes (pH 4-5) and the presence of chloride ions inside the cell.
Figure 16 shows the hematological parameters for treatment of mice with 40mg/kg and 80mg/kg liposomal aG6 administered once per week for 4 weeks: effects of White Blood Cell (WBC) count, neutrophil count, and platelet count. No significant decrease in mean neutrophil, mean leukocyte or mean platelet counts was observed.
FIG. 17 shows the effect of treatment of mice with 40mg/kg and 80mg/kg liposomal aG6 administered once per week for 4 weeks on hemoglobin and reticulocyte indices. At higher dose levels, the mean hemoglobin concentration decreases minimally. In parallel, the mean reticulocyte proliferation index increased slightly.
Figure 18 shows the effect of treatment of mice with 40mg/kg and 80mg/kg liposomal aG6 administered once per week for 4 weeks on liver markers including serum aspartate Aminotransferase (AST) and serum alanine Aminotransferase (ALT) and serum albumin. There was no significant increase in mean AST or mean ALT levels of hepatic transaminase, and there was no observed change in mean albumin levels.
Figure 19 shows the relative tumor volumes of immunodeficient female Nu/J mice (6-8 weeks old) inoculated with NCI-H292 (non-small cell lung cancer) cells and intravenously administered 167mg/kg once every three weeks, control, pemetrexed and liposomal aG 6. As can be seen from these preliminary data, liposomal aG6 provides reduced tumor control compared to pemetrexed.
Figures 20A-20F show dose response relationships of liposomal pemetrexed α -L triglutamate (liposomal aG3), liposomal pemetrexed α -L pentaglutamate (liposomal aG5), liposomal pemetrexed α -L octaglutamate (liposomal aG7), and a combination of liposomal pemetrexed α -L hexaglutamate (aG6) and α -L dodecaglutamate (aG12) (liposomal aG6 and aG12) to H2342(NSCLC, adenocarcinoma subtype) (figure 20A), H292(NSCLC, adenocarcinoma subtype) (figure 20B), HT-29 (colon cancer) (figure 20C), HCC1806 (triple negative breast cancer) (figure 20D), MCF7(ER + breast cancer) (figure 20E), and OAW28 (ovarian cancer) (figure 20F) within 48 hours. Substantially as described in example 1, by
Figure BDA0002715283060000181
(CTG) luminescence cell viability assay to determine cell viability. As shown in all cell lines, the efficacy of each polyglutamated pemetrexed liposome composition greatly exceeded that of the liposome vehicle and empty liposome controls.
Detailed Description
The present disclosure relates generally to novel alpha polyglutamated methotrexate compositions. The compositions provide progression over prior treatments for hyperproliferative diseases, such as cancer. Methods of making, delivering, and using the alpha-polyglutamated methotrexate compositions are also provided. The alpha polyglutamated compositions have uses including, but not limited to, the treatment or prevention of hyperproliferative diseases (such as cancer), immune system disorders (such as rheumatoid arthritis), and infectious diseases (such as HIV and malaria).
I. Definition of
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.
It should be understood that when the language "comprises" is used herein to describe embodiments, other similar embodiments described in terms of "comprising," "consisting of … …," and/or "consisting essentially of … …" are also provided. However, when used as a transitional phrase in a claim, each phrase should be interpreted separately and in the appropriate legal and factual context (e.g., in the claims, the transitional phrase "comprising" is more considered an open phrase than "consisting of … …" is more exclusive, and "consisting essentially of … …" takes an intermediate position).
As used herein, the singular forms "a", "an" and "the" include plural referents unless the content clearly dictates otherwise or is clear from the context.
The term "and/or" as used in phrases such as "a and/or B" herein is intended to include a and B; a or B; a (alone) and B (alone). Also, the term "and/or" as used in phrases such as "A, B and/or C" is intended to encompass each of the following embodiments: A. b and C; A. b or C; a or C; a or B; b or C; a and C; a and B; b and C; a (alone); b (alone); and C (alone).
Unless otherwise indicated, the terms "methotrexate" and "MTX" are used interchangeably to include salts, acids, and/or free base forms of methotrexate (e.g., disodium methotrexate). The compositions containing the MTX salt may also contain any of a variety of cations, such as Na+、Mg2+、K+、NH4 +And/or Ca2+. In certain embodiments, the salt is a pharmaceutically acceptable salt. In another specific embodiment, the MTX salt contains Na+. Methotrexate contains one L-gamma glutamyl group and is therefore considered monoglutamated for the purposes of this disclosure.
The headings and sub-headings are used for convenience and/or form compliance only, do not limit the subject technology, and are not mentioned in connection with the explanation of the description of the subject technology. In various embodiments, features described under one heading or one subheading of the disclosure may be combined with features described under other headings or subheadings. Furthermore, not all features under a single title or a single subtitle may necessarily be used together in an embodiment.
The terms "polyglutamate", "polyglutamation", or variations thereof refer to a composition comprising at least one chain of 2 or more linked glutamyl groups. The polyglutamate chain may be linear or branched. The linear polyglutamate chain may comprise, for example, glutamyl groups containing alpha carboxyl or gamma carboxyl linkages. The branched polyglutamate chain may comprise, for example, one or more glutamyl groups containing linkages to the alpha and gamma carboxyl groups of other glutamyl groups, thereby providing branch points for the polyglutamate. Exemplary branched polyglutamates are shown in FIG. 1R-1U. The polyglutamate chain comprises an N-terminal glutamyl group and one or more C-terminal glutamyl groups. The N-terminal glutamyl group of the polyglutamate chain is not linked to another glutamyl group via its amine group, but is linked to one or more glutamyl groups via its carboxylic acid group. In some embodiments, the N-terminal glutamyl group of the polyglutamated methotrexate is a glutamyl group of methotrexate. One or more C-terminal glutamyl groups of the polyglutamate chain are linked to another glutamyl group via its amine group, but not via its carboxylic acid group.
The terms "polyglutamated methotrexate," "polyglutamated MTX," "MTX-PG," "PMTX," and iterative forms thereof are used interchangeably herein to refer to a methotrexate composition that includes at least one glutamyl group in addition to the glutamyl group of methotrexate (i.e., MTX-PG)nWherein n is more than or equal to 1). The glutamyl group of methotrexate is taken into account when referring herein to the number of glutamyl groups in alpha PMTX (MTX-PG). For example, an MTX-PG composition that contains 5 glutamyl residues in addition to the glutamyl residues of MTX is referred to herein as methotrexate hexaglutaminate or methotrexate hexaglutamate.
The terms "α glutamyl", "α glutamate" and "α linkage" as they relate to the linkage of a glutamyl group refer to a glutamyl group containing an α carboxyl linkage. In some embodiments, the alpha linkage is an amide linkage between the alpha carboxyl group of one glutamyl group and a second glutamyl group. The alpha linkage can be between glutamyl and the glutamyl group of methotrexate, or between the glutamyl group and a second glutamyl group not present in methotrexate (such as the glutamyl group in the polyglutamate chain to which methotrexate is attached).
The terms "gamma glutamyl", "gamma glutamate" and "gamma linkage" as they relate to the linkage of a glutamyl group refer to a glutamyl group containing a gamma carboxyl linkage. As described herein, once methotrexate enters the cell, it is polyglutamated by the enzyme folate poly-gamma-glutamate synthase (FPGS), which continuously adds L-glutamyl groups to the gamma carboxyl groups of glutamate within methotrexate. Thus, during methotrexate treatment, no α -polyglutamated methotrexate compositions are formed intracellularly. In some embodiments, the gamma linkage is an amide linkage between the gamma carboxyl group of one glutamyl group and a second glutamyl group. The gamma linkage can be between glutamyl and glutamyl of methotrexate, or between glutamyl and a second glutamyl not present in methotrexate (such as glutamyl in the polyglutamate chain linked to methotrexate). In some embodiments, the γ linkage refers to the amide bond of glutamyl in methotrexate. Unless explicitly indicated or clearly understood from the context that this is not intended, reference to a gamma linkage includes the gamma linkage of a glutamyl group in methotrexate.
Unless otherwise indicated, the terms "α polyglutamated methotrexate," "α PMTX," "α -MTX-PG," and iterative forms thereof, are used interchangeably herein to refer to polyglutamated methotrexate compositions comprising at least one α -linked glutamyl group. For example, for the purposes of this disclosure, a pentaglutamized-MTX composition in which the 2 nd glutamyl group has an alpha linkage, but each other glutamyl group has a gamma linkage, is considered to be an alpha-MTX-PG. In some embodiments, each glutamyl group of MTX-PG has an alpha linkage in addition to the glutamyl group of MTX (e.g., MTX-PG)nWherein n is 5, and wherein G1、G2、G3、G4And G5Each having an alpha linkage). In some embodiments, each glutamyl group of MTX-PG has, in addition to one or more C-terminal glutamyl groups and a glutamyl group of MTXWith alpha linkage (e.g. MTX-PG)nWherein n is 5, and wherein G1、G2、G3And G4Each having an alpha linkage). In some embodiments, each glutamyl group of the MTX-PG has an alpha linkage in addition to one or more C-terminal glutamyl groups (e.g., MTX-PG)nWherein n is 5, and wherein glutamyl of MTX and G1、G2、G3And G4Each having an alpha linkage).
As used herein, the term "isolated" refers to a composition in a form not found in nature. Isolated alpha polyglutamated compositions include those that have been purified to the extent that they are no longer in the form in which they are found in nature. In some embodiments, the isolated alpha polyglutamated methotrexate is substantially pure. Isolated compositions will be free or substantially free of materials with which they are naturally associated, such as other cellular components, such as proteins and nucleic acids, that may be present with them in nature or their environment of preparation (e.g., cell culture). The alpha polyglutamated composition may be formulated with diluents or adjuvants and still be isolated for practical purposes-for example, when used in diagnosis or therapy, the alpha polyglutamated composition will typically be mixed with a pharmaceutically acceptable carrier or diluent. In some embodiments, an isolated α polyglutamate composition (e.g., α polyglutamate and a delivery vehicle, such as a liposome containing α polyglutamate, contains less than 1% or less than 0.1% of undesirable DNA or protein content.
The term "targeting moiety" is used herein to refer to a molecule that provides enhanced affinity for a selected target (e.g., a cell, cell type, tissue, organ, region or compartment of the body, such as a cell, tissue or organ compartment). The targeting moiety may comprise a wide variety of entities. The targeting moiety may comprise a naturally occurring molecule, or a recombinant or synthetic molecule. In some embodiments, the targeting moiety is an antibody, antigen-binding antibody fragment, bispecific antibody, or other antibody-based molecule or compound. In some embodiments, the targeting moiety is an aptamer, an avimer, a receptor binding ligand, a nucleic acid, a biotin-avidin binding pair, a peptide, a protein, a carbohydrate, a lipid, a vitamin, a toxin, a component of a microorganism, a hormone, a receptor ligand, or any derivative thereof. Other targeting moieties are known in the art and are encompassed by the present disclosure.
The term "specific affinity" or "specifically binds" refers to some combination of more frequent, faster, longer lasting, greater affinity, or the like, reaction or association of a targeting moiety (e.g., an antibody or antigen-binding antibody fragment) with an epitope, protein, or target molecule as compared to a surrogate (including a protein not associated with the epitope of interest). Due to sequence identity between homologous proteins in different species, in some embodiments, specific affinity may include a binding agent that recognizes a protein or target in more than one species. Likewise, due to homology within certain regions of the polypeptide sequences of different proteins, the terms "specific affinity" or "specific binding" may include binders that recognize more than one protein or target. It will be appreciated that in certain embodiments, a targeting moiety that specifically binds a first target may or may not specifically bind a second target. Thus, "specific affinity" does not necessarily require (although may include) unique binding, e.g., to a single target. Thus, in certain embodiments, a targeting moiety may specifically bind more than one target. In certain embodiments, multiple targets may be bound by the same targeting moiety.
The term "epitope" refers to that portion of an antigen that is capable of being recognized and specifically bound by a targeting moiety (i.e., a binding moiety), such as an antibody. When the antigen is a polypeptide, the epitope may be formed by contiguous amino acids and non-contiguous amino acids juxtaposed by tertiary folding of the protein. Epitopes formed by contiguous amino acids are typically retained when the protein is denatured, while epitopes formed by tertiary folding are typically lost when the protein is denatured. Epitopes typically comprise at least 3, and more typically at least 5 or 8-10 amino acids in a unique spatial conformation.
Expressions such as "binding affinity to a target", "binding to a target" and similar expressions known in the art refer to the properties of a targeting moiety that can be directly measured by determining the affinity constant, e.g., the amount of the targeting moiety that associates and dissociates at a given antigen concentration. Different methods can be used to characterize molecular interactions, such as, but not limited to, competition assays, equilibrium assays, and microcalorimetric assays, as well as real-time interaction assays based on surface plasmon resonance interactions (e.g., using
Figure BDA0002715283060000211
Instrument). These methods are well known to those skilled in the art and are described, for example, in Neri et al, Tibtech 14: 465-.
The term "delivery vehicle" generally refers to any composition used to assist, facilitate or assist the entry of alpha-polyglutamated methotrexate into a cell. Such delivery vehicles are known in the art and include, but are not limited to, liposomes, lipid spheres, polymers (e.g., polymer-conjugates), peptides, proteins such as antibodies (e.g., immunoconjugates, such as Antibody Drug Conjugates (ADCs)) and antigen-binding antibody fragments and their derivatives), cellular components, cyclic oligosaccharides (e.g., cyclodextrins), micelles, microparticles (e.g., microspheres), nanoparticles (e.g., lipid nanoparticles, biodegradable nanoparticles, and core-shell nanoparticles), hydrogels, lipoprotein particles, viral sequences, viral materials, or lipid or liposome formulations, and combinations thereof. The delivery vehicle may be directly or indirectly linked to the targeting moiety. In some examples, the targeting moiety is selected from a macromolecule, a protein, a peptide, a monoclonal antibody, or a fatty acid lipid.
By "subject" is meant a human or vertebrate mammal, including but not limited to dogs, cats, horses, goats, and primates, such as monkeys. Thus, the invention may also be used to treat a disease or disorder in a non-human subject. For example, cancer is one of the leading causes of death in companion animals (i.e., cats and dogs). In some embodiments of the invention, the subject is a human. In the present disclosure, the terms "subject" and "patient" are used interchangeably and have the same meaning. It is generally preferred to use the maximum dose, i.e., the highest safe dose according to sound medical judgment.
As used herein, "effective amount" refers to a dose of an agent sufficient to provide a medically desirable result. The effective amount will vary with the desired result, the particular condition being treated or prevented, the age and physical condition of the subject being treated, the severity of the condition, the duration of the treatment, the nature of concurrent or combined therapy (if any), the particular route of administration, and like factors within the knowledge and expertise of a health practitioner. For the purposes set forth, an effective amount can be determined empirically and in a conventional manner. In the case of cancer, an effective amount of the agent can reduce the number of cancer cells; reducing the size of the tumor; inhibit (i.e., slow to some extent and preferably prevent) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and preferably prevent) tumor metastasis; inhibit tumor growth to some extent; and/or alleviate one or more symptoms associated with the condition to some extent. To the extent the drug can prevent growth and/or kill existing cancer cells, it can be cytostatic and/or cytotoxic. For cancer therapy, in vivo efficacy can be measured, for example, by assessing duration of survival, duration of progression-free survival (PFS), response rate (e.g., RR), response duration, and/or quality of life.
The terms "hyperproliferative disorder", "proliferative disease" and "proliferative disorder" are used interchangeably herein and relate to unwanted or uncontrolled cellular proliferation of excessive or abnormal cells, which is not desired, such as tumors or proliferative growth (whether in vitro or in vivo). In some embodiments, the proliferative disease is a cancer or a tumor disease (including benign or cancerous) and/or any metastasis, regardless of where the cancer, tumor and/or metastasis is located. In some embodiments, the proliferative disease is a benign or malignant tumor. In some embodiments, the proliferative disease is a non-cancerous disease. In some embodiments, the proliferative disease is a hyperproliferative disorder, such as hyperplasia, fibrosis (especially pulmonary fibrosis, but also including other types of fibrosis, such as renal fibrosis), angiogenesis, psoriasis, atherosclerosis, and smooth muscle proliferation in blood vessels, such as stenosis or restenosis following angioplasty.
"cancer," "tumor," or "malignant disease" are used synonymously and refer to any of a variety of diseases characterized by uncontrolled, abnormal proliferation of cells, the ability of affected cells to spread locally or through the bloodstream and lymphatic system to other parts of the body (metastasis), and any of a number of characteristic structural and/or molecular features. As used herein, "tumor" refers to all neoplastic cell growth and proliferation (whether malignant or benign) as well as all precancerous and cancerous cells and tissues. By "cancerous tumor" or "malignant cell" is understood a cell that has specific structural properties, lacks differentiation and is capable of invading and metastasizing. Cancers that can be treated using the alpha PMTX compositions provided herein include, but are not limited to, non-hematologic malignancies, including, for example, lung cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer, biliary tract cancer, gallbladder cancer, bladder cancer, sarcomas (e.g., osteosarcoma), brain cancer, central nervous system cancer, and melanoma; and hematological malignancies such as leukemia, lymphoma and other B cell malignancies, myeloma and other plasma cell dysplasias or dyscrasias. Other types of cancers and tumors that can be treated using the alpha PMTX compositions are described herein or are otherwise known in the art. The terms "cancer," "cancerous," "cell proliferative disorder," "proliferative disorder," and "tumor" are not mutually exclusive when referred to herein.
Terms such as "treating" or "treatment" or "treating" refer to (a) therapeutic measures to cure, slow down, alleviate symptoms of, and/or halt the progression of the diagnosed pathological condition or disorder and (b) prophylactic or preventative measures to prevent and/or slow the development of the targeted disease or condition. Thus, a subject in need of treatment includes a subject already suffering from a cancer, disorder or disease; those subjects at risk for cancer or a condition; and those subjects in whom the infection or condition is to be prevented. Using well known medical and diagnostic techniques, a subject is identified as "having" or at risk of having a cancer, an infectious disease, an immune system disorder, a hyperproliferative disease, or another disease or disorder mentioned herein. In certain embodiments, a subject is successfully "treated" according to the methods provided herein if the subject exhibits a full, partial, or transient improvement or elimination of, for example, symptoms associated with a disease or disorder (e.g., cancer, rheumatoid arthritis). In particular embodiments, the terms "treating" or "treatment" refer to ameliorating at least one measurable physical parameter of a proliferative disorder, such as tumor growth, which is not necessarily discernible by the patient. In other embodiments, the terms "treating" or "treatment" refer to inhibiting the progression of a proliferative disorder in the body, for example, by stabilizing a discernible symptom or physiologically, for example, by stabilizing a physical parameter, or both. In other embodiments, the term "treating" or "treatment" refers to a reduction or stabilization of tumor size, tumor cell proliferation or survival, or cancer cell count. The alpha-PMTX compositions can be used alone or in combination with additional therapeutic agents for treatment.
"subject" and "patient" and "animal" are used interchangeably and refer to mammals such as human patients and non-human primates as well as laboratory animals such as rabbits, rats, and mice, among others. Animals include all vertebrates, e.g., mammals and non-mammals, such as chickens, amphibians, and reptiles. As used herein, "mammal" refers to any member of the class mammalia, including, but not limited to, humans and non-human primates, such as chimpanzees, and other apes and monkey species; livestock such as cattle, sheep, pigs, goats, and horses; domestic mammals, such as dogs and cats; laboratory animals, including rodents, such as mice, rats and guinea pigs, and other members of the class mammalia known in the art. In a particular embodiment, the patient is a human.
"treatment of a proliferative disorder" is used herein to include maintaining or reducing tumor size, inducing tumor regression (partial or complete), inhibiting tumor growth, and/or increasing the lifespan of a subject having a proliferative disorder. In one embodiment, the proliferative disorder is a solid tumor. Such tumors include, for example, lung cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, renal cancer, cholangiocarcinoma, gallbladder cancer, bladder cancer, sarcoma (e.g., osteosarcoma), brain cancer, central nervous system cancer, and melanoma. In one embodiment, the proliferative disorder is a hematologic malignancy. Such hematological malignancies include, for example, leukemias, lymphomas and other B cell malignancies, myelomas and other plasma cell dysplasias or dyscrasias. In some embodiments, the cancer is selected from the group consisting of: breast cancer, head and neck cancer, lung cancer, gastric cancer, osteosarcoma, non-hodgkin lymphoma (NHL), Acute Lymphoblastic Leukemia (ALL), mycosis fungoides (cutaneous T-cell lymphoma), choriocarcinoma and chorioadenoma, non-leukemic meningeal cancer, soft tissue sarcoma (desmoid, invasive fibromatosis), bladder cancer, and Central Nervous System (CNS) lymphoma.
The term "autoimmune disease" as used herein is defined as a condition caused by an autoimmune response. Autoimmune diseases are the result of inappropriate and excessive responses to self-antigens. Examples of autoimmune diseases include, but are not limited to, Addison's disease, alopecia areata, ankylosing spondylitis, autoimmune hepatitis, autoimmune mumps, Crohn's disease, diabetes (type I), dystrophic bullous epidermolysis, epididymitis, glomerulonephritis, Graves ' disease, Guillain-Barre syndrome, Hashimoto's disease, hemolytic anemia, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, spondyloarthropathies, thyroiditis, vasculitis, vitiligo, myxoedema, pernicious anemia, ulcerative colitis, and the like.
The term "therapeutic agent" is used herein to refer to an agent or derivative thereof that interacts with a hyperproliferative cell, such as a cancer cell or an immune cell, to reduce the proliferative state of said cell and/or kill said cell. Examples of therapeutic agents include, but are not limited to, chemotherapeutic agents, cytotoxic agents, platinum-based agents (e.g., cisplatin, carboplatin, oxaliplatin), taxanes (e.g.,
Figure BDA0002715283060000221
l), etoposide, alkylating agents (e.g., cyclophosphamide, ifosfamide), metabolic antagonists (e.g., Methotrexate (MTX), 5-fluorouracil gemcitabine or derivatives thereof), antitumor antibiotics (e.g., mitomycin, doxorubicin), plant-derived antitumor agents (e.g., vincristine, vindesine, cyclophosphamide, and the like), and pharmaceutically acceptable salts thereof,
Figure BDA0002715283060000222
l). Such agents may also include, but are not limited to, the anticancer agents trimetrexate, temozolomide, raltitrexed, S- (4-nitrobenzyl) -6-thioinosine (NBMPR), 6-benzylguanidine (6-BG), bis-chloronitrosourea (BCNU), and CAMPTOTHECINTMOr a therapeutic derivative of either of them. Additional examples of therapeutic agents that may be suitable for use in accordance with the disclosed methods include, but are not limited to, anti-restenotic agents, pro-proliferative or anti-proliferative agents, anti-inflammatory agents, anti-neoplastic agents, anti-mitotic agents, anti-platelet agents, anti-coagulant agents, anti-fibrin agents, anti-thrombin agents, cytostatic agents, antibiotics and other anti-infective agents, anti-thrombin agents, anti-metabolite agents, angiogenic agents, cytoprotective agents, Angiotensin Converting Enzyme (ACE) inhibitors, angiotensin II receptor antagonists, and/or cardioprotective agents. "therapeutic agent" also refers to salts, acids, and free base forms of the above agents.
As used herein, the term "chemotherapeutic agent," when used in relation to cancer therapy, refers to any agent that causes cancer cells to die or inhibits the growth or spread of cancer cells. Examples of such chemotherapeutic agents include alkylating agents, antibiotics, antimetabolites, plant derived agents, and hormones. In some embodiments, the chemotherapeutic agent is cisplatin. In some embodiments, the chemotherapeutic agent is carboplatin. In some embodiments, the chemotherapeutic agent is oxaliplatin. In other embodiments, the chemotherapeutic agent is gemcitabine. In other embodiments, the chemotherapeutic agent is doxorubicin.
The term "antimetabolite" is used herein to refer to therapeutic agents that inhibit the utilization of metabolites or prodrugs thereof. Examples of antimetabolites include methotrexate, pemetrexed, 5-fluorouracil prodrugs such as capecitabine, 5-fluorodeoxyuridine monophosphate, cytarabine prodrugs such as nelarabine, 5-azacytidine, gemcitabine, mercaptopurine, thioguanine, azathioprine, adenosine, pentostatin, erythrohydroxynonyladenine (erythrohydroxynonyladenine), and cladribine. Antimetabolites useful for practicing the methods of the present disclosure include nucleoside analogs, including purine or pyrimidine analogs. In some embodiments, the alpha polyglutamated methotrexate composition is used in combination with an antimetabolite selected from the group consisting of: fluoropyrimidine 5-fluorouracil, 5-fluoro-2' -deoxycytidine, cytarabine, gemcitabine, troxacitabine, decitabine, azacytidine, pseudoisocytidine, zebularine, ancitabine, fazarabine, 6-azacytidine, capecitabine, N 4-octadecyl-cytarabine, cytarabine elaidate, fludarabine, cladribine, clofarabine, nelarabine, forodesine (forodesine) and pentostatin or their derivatives. In one example, a nucleoside analog is a substrate for a nucleoside deaminase, which is either adenosine deaminase or cytidine deaminase. In some examples, the nucleoside analog is selected from fludarabine, cytarabine, gemcitabine, decitabine, and azacytidine, or derivatives thereof. In certain embodiments, the antimetabolite is 5-fluorouracil.
As used herein, a "taxane" is an interfering or disrupting speciesAnticancer agents that stabilize, form and/or function microtubules. Taxane agents include paclitaxel and docetaxel and their derivatives, which act on microtubules in the same way as the taxane from which they are derived. In certain embodiments, the taxane is paclitaxel or docetaxel, or a pharmaceutically acceptable salt, acid, or derivative of paclitaxel or docetaxel. In certain embodiments, the taxane is paclitaxel
Figure BDA0002715283060000231
Docetaxel
Figure BDA0002715283060000232
Albumin-bound paclitaxel (nano albumin-bound paclitaxel;
Figure BDA0002715283060000233
) DHA-paclitaxel or PG-paclitaxel.
The terms "pharmaceutically acceptable carrier" and "pharmaceutically acceptable carrier" refer to ingredients of a pharmaceutical formulation other than an active ingredient that are not toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, carriers, excipients, stabilizers, diluents, or preservatives. A pharmaceutically acceptable carrier may include, for example, one or more compatible solid or liquid fillers, diluents, or encapsulating substances suitable for administration to a human or other subject.
The present disclosure relates generally to novel alpha polyglutamated Methotrexate (MTX) compositions, as well as methods of making and using the compositions to treat diseases, including hyperproliferative diseases (such as cancer), immune system disorders (such as rheumatoid arthritis), and infectious diseases (such as HIV and malaria).
In some embodiments, the present disclosure provides:
[1] a composition comprising alpha polyglutamated methotrexate, wherein at least one glutamyl group has an alpha carboxyl linkage;
[2] the composition of [1], wherein the alpha polyglutamated methotrexate comprises 1-10 glutamyl groups having an alpha carboxy linkage;
[3] The composition of any of [1] to [2], wherein the alpha-polyglutamated methotrexate contains 4, 5, 6, 2-10, 4-6, or more than 5 glutamyl groups;
[4] the composition of any of [1] to [3], comprising alpha tetraglutamated methotrexate;
[5] the composition of any one of [1] to [3], comprising alpha-pentaglutamated methotrexate;
[6] the composition of any one of [1] to [3], comprising alpha-hexaglutaminated methotrexate;
[7] the composition according to any one of [1] to [6], wherein
(a) Two or more glutamyl groups have an alpha carboxyl linkage,
(b) each glutamyl group, other than the glutamyl group of methotrexate, has an alpha carboxyl linkage; or
(c) Two or more glutamyl groups have a gamma carboxyl linkage,
[8] the composition according to any one of claims 1 to 6, wherein
(a) Each glutamyl group, except for one or more C-terminal glutamyl groups and the glutamyl group of methotrexate, has an alpha carboxyl linkage; or
(b) Each glutamyl group other than the one or more C-terminal glutamyl groups has an alpha carboxyl linkage;
[9] the composition of any one of [1] to [8], wherein at least one glutamyl group has both an alpha carboxyl linkage and a gamma carboxyl linkage;
[10] The composition according to any one of [1] to [9], wherein:
(a) at least 2 glutamyl groups of the alpha polyglutamated methotrexate are in the L form,
(b) each glutamyl group of the alpha polyglutamated methotrexate is in the L form,
(c) at least 1 glutamyl group of said alpha polyglutamated methotrexate is in the D form,
(d) each glutamyl group of said alpha polyglutamated methotrexate is in the D form, in addition to the glutamyl group of methotrexate, or
(e) At least 2 glutamyl groups of the alpha polyglutamated methotrexate are in the L form, and at least 1 glutamyl group is in the D form;
[11] the composition of any one of [1] to [10], wherein the polyglutamate is linear;
[12] the composition of any one of [1] to [10], wherein the polyglutamate is branched;
[13] a liposome composition comprising alpha polyglutamated methotrexate (Lp-alpha PMTX) according to any one of [1] to [12 ];
[14] the L α PP composition of [13], wherein the α polyglutamated methotrexate comprises glutamyl in L form having an α carboxyl linkage;
[15] the Lp- α PMTX composition according to [13] or [14], wherein each glutamyl group of the α polyglutamated methotrexate is in the L form;
[16] The Lp- α PMTX composition of [13] or [14], wherein at least one glutamyl group of the α polyglutamated methotrexate is in the D form;
[17] the Lp-alpha PMTX composition of any of [13] - [16], wherein the liposome comprises alpha polyglutamated methotrexate containing 4, 5, 2-10, 4-6, or more than 5 glutamyl groups;
[18] the Lp- α PMTX composition of any one of [13] - [17], wherein at least one glutamyl group of the α polyglutamated methotrexate has a γ carboxyl linkage;
[19] the composition of any one of [13] to [18], wherein at least one glutamyl group has both an alpha carboxyl linkage and a gamma carboxyl linkage;
[20] the composition of any one of [13] to [19], the composition containing 2, 3, 4, 5, 2-10, 4-6, or more than 5 glutamyl groups having both alpha and gamma carboxyl linkages;
[21] the Lp-alpha PMTX composition of any of [13] - [20], wherein the liposome comprises alpha polyglutamated methotrexate that contains alpha tetraglutamated methotrexate, alpha pentaglutamated methotrexate, or alpha hexaglutamated methotrexate;
[22] The Lp-alpha PMTX composition of any one of [13] - [21], wherein the polyglutamate is linear or branched;
[23] the Lp- α PMTX composition of any one of [13] - [22], wherein the liposome is pegylated (pa Lp- α PMTX);
[24] the Lp-alpha PMTX composition of any one of [13] - [23], wherein the liposomes comprise at least 1% weight/weight (w/w) of the alpha polyglutamated methotrexate, or wherein at least 1% of the starting material of alpha polyglutamated MTX is encapsulated (embedded) in the alpha PMTX during the process of preparing the Lp-alpha PMTX;
[25] the Lp- α PMTX composition of any one of [13] - [24], wherein the liposome has a diameter in the range of 20nm to 500nm or 20nm to 200 nm;
[26] the Lp- α PMTX composition of any one of [13] - [25], wherein the liposome has a diameter in the range of 80nm to 120 nm;
[27] the Lp-alpha PMTX composition of any one of [13] - [26], wherein the liposome is formed from liposome components;
[28] the Lp-alpha PMTX composition of [27], wherein the liposomal composition comprises at least one of anionic lipids and neutral lipids;
[29] The Lp- α PMTX composition according to [27] or [28], wherein the liposome component comprises at least one selected from the group consisting of: DSPE; DSPE-PEG; DSPE-PEG-maleimide; HSPC; HSPC-PEG; cholesterol; cholesterol-PEG; and cholesterol-maleimide.
[30] The Lp-alpha PMTX composition of any one of [27] - [29], wherein the liposome component comprises at least one selected from the group consisting of: DSPE; DSPE-PEG; DSPE-PEG-FITC; DSPE-PEG-maleimide; cholesterol; and HSPC;
[31] the Lp- α PMTX composition of any one of [27] to [30], wherein one or more liposomal components further comprises a steric stabilizer;
[32] the Lp- α PMTX composition of [31], wherein the steric stabilizer is at least one selected from the group consisting of: polyethylene glycol (PEG); poly-L-lysine (PLL); monosialoganglioside (GM 1); poly (vinyl pyrrolidone) (PVP); poly (acrylamide) (PAA); poly (2-methyl-2-oxazoline); poly (2-ethyl-2-oxazoline); a phosphatidylpolyglycerol; poly [ N- (2-hydroxypropyl) methacrylamide ]; amphiphilic poly-N-vinylpyrrolidone; an L-amino acid-based polymer; oligomerization of glycerol; copolymers comprising polyethylene glycol and polypropylene oxide; poloxamer 188; and polyvinyl alcohol;
[33] The Lp- α PMTX composition of [32], wherein the steric stabilizer is PEG, and the PEG has a number average molecular weight (Mn) of 200 to 5000 daltons;
[34] the Lp-alpha PMTX composition of any one of [13] to [33], wherein the liposome is anionic or neutral;
[35] the Lp-alpha PMTX composition of any of [13] - [33], wherein the liposome has a zeta potential less than or equal to zero;
[36] the Lp- α PMTX composition of any of [13] - [33], wherein the liposome has a zeta potential between 0 to-150 mV;
[37] the Lp-alpha PMTX composition of any of [13] - [33], wherein the liposome has a zeta potential between-30 to-50 mV;
[38] the Lp- α PMTX composition of any one of [13] to [33], wherein the liposome is cationic;
[39] the Lp-alpha PMTX composition of any one of [13] - [38], wherein the liposome has an interior space comprising the alpha polyglutamated methotrexate and a pharmaceutically acceptable aqueous carrier;
[40] the Lp- α PMTX composition of [39], wherein the pharmaceutically acceptable carrier comprises a tonicity agent such as dextrose, mannitol, glycerol, potassium chloride, sodium chloride at a concentration of greater than 1%;
[41] The Lp- α PMTX composition of [39], wherein the pharmaceutically acceptable aqueous carrier is trehalose;
[42] the Lp- α PMTX composition of [41], wherein the pharmaceutically acceptable carrier comprises 5 to 20% trehalose by weight;
[43] the Lp-alpha PMTX composition of any one of [39] - [42], wherein the pharmaceutically acceptable carrier comprises from 1% to 15% by weight of dextrose;
[44] the Lp- α PMTX composition of any of [39] - [43], wherein the interior space of the liposome comprises 5% dextrose suspended in HEPES buffer solution;
[45] the Lp-alpha PMTX composition of any one of [39] - [44], wherein the pharmaceutically acceptable carrier comprises a buffer, such as HEPES Buffered Saline (HBS) or the like, at a concentration of between 1 and 200mM and a pH of between 2 and 8;
[46] the Lp-alpha PMTX composition of any one of [39] - [45], wherein the pharmaceutically acceptable carrier comprises sodium acetate and calcium acetate in a total concentration between 50mM to 500 mM;
[47] the Lp- α PMTX composition of any one of [13] - [46], wherein the interior space of the liposome has a pH of 5-8 or a pH of 6-7, or any range therebetween;
[48] The Lp-alpha PMTX composition of any one of [13] - [47], wherein the liposome comprises less than 500,000 or less than 200,000 of the alpha polyglutamated methotrexate molecules;
[49] the Lp-alpha PMTX composition of any of [13] - [48], wherein the liposome comprises between 10 to 100,000 of the alpha polyglutamated methotrexate molecules, or any range therebetween;
[50] the Lp-alpha PMTX composition of any one of [13] - [49], further comprising a targeting moiety, and wherein the targeting moiety has specific affinity for a surface antigen on a target cell of interest;
[51] the Lp-a PMTX composition of [50], wherein the targeting moiety is attached to one or both of PEG and the exterior of the liposome, optionally wherein targeting moiety is attached to one or both of the PEG and the exterior of the liposome by a covalent bond;
[52] the Lp- α PMTX composition of [50] or [51], wherein the targeting moiety is a polypeptide;
[53] the Lp- α PMTX composition of any one of [50] - [52], wherein the targeting moiety is an antibody or an antigen-binding fragment of an antibody;
[54]according to [50 ]-[53]The Lp-alpha PMTX composition of any one of, wherein, if used
Figure BDA0002715283060000251
The targeting moiety is measured by analysis to be at 0.5x 10-10To 10x 10-6An equilibrium dissociation constant (Kd) in a range binds to the surface antigen;
[55] the Lp-alpha PMTX composition of any one of [50] - [55], wherein the targeting moiety specifically binds to one or more folate receptors selected from the group consisting of: folate receptor alpha (FR-alpha), folate receptor beta (FR-beta), and folate receptor (FR-);
[56] the Lp-alpha PMTX composition of any one of [50] - [56], wherein the targeting moiety comprises one or more selected from the group consisting of: antibodies, humanized antibodies, antigen-binding fragments of antibodies, single chain antibodies, single domain antibodies, bispecific antibodies, synthetic antibodies, pegylated antibodies, and multimeric antibodies;
[57] the Lp- α PMTX composition of any one of [50] - [56], wherein each pegylated liposome comprises 1 to 1000 or 30 to 200 targeting moieties;
[58] the Lp-alpha PMTX composition of any one of [39] - [57], further comprising one or more of an immunostimulatory agent, a detectable label, and a maleimide, wherein the immunostimulatory agent, the detectable label, or the maleimide is attached to the exterior of the PEG or the liposome;
[59] The Lp- α PMTX composition of [58], wherein the immunostimulatory agent is at least one selected from the group consisting of: a protein immunostimulant; a nucleic acid immunostimulant; a chemical immunostimulant; a hapten; and an adjuvant;
[60]such as [58]]Or [59]]The Lp-alpha PMTX composition, wherein the immunostimulatory agent is at least one selected from the group consisting of: fluorescein; fluorescein Isothiocyanate (FITC); DNP; beta glucan; beta-1, 3-glucan; beta-1, 6-glucan; resolvins (e.g., resolvins D such as D)n-6DPAOr Dn-3DPAResolvin E, or T series resolvin); and Toll-like receptor (TLR) modulators, such as oxidized low density lipoproteins (e.g., OXPAC, PGPC) and eritoran lipids (e.g., E5564);
[61] the Lp- α PMTX composition of any one of [58] to [60], wherein the immunostimulatory agent and the detectable label are the same;
[62] the Lp-alpha PMTX composition of any one of [58] to [61], further comprising a hapten;
[63] the Lp- α PMTX composition of [62], wherein the hapten comprises one or more of fluorescein or β 1, 6-glucan;
[64] the Lp-alpha PMTX composition of any of [13] - [63], further comprising in the interior space, the exterior space, or both the interior space and the exterior space at least one cryoprotectant selected from the group consisting of: mannitol; trehalose; sorbitol; and sucrose;
[65] A targeting composition comprising the composition according to any one of [1] to [64 ];
[66] a non-targeted composition comprising the composition according to any one of [1] to [49 ];
[67] the Lp-alpha PMTX composition of any one of [13] - [66], further comprising carboplatin and/or pembrolizumab;
[68] a pharmaceutical composition comprising a liposomal alpha-polyglutamated methotrexate composition according to any one of [13] to [67 ];
[69] a pharmaceutical composition comprising an alpha polyglutamated methotrexate composition according to any one of [1] to [8 ];
[70] a composition as described in any one of [1] to [69], for use in the treatment of a disease;
[71] use of a composition as defined in any one of [1] to [70] in the manufacture of a medicament for the treatment of a disease;
[72] a method for treating or preventing a disease in a subject in need of such treatment or prevention, the method comprising administering to the subject a composition as described in any one of [1] to [70 ];
[73] a method for treating or preventing a disease in a subject in need of such treatment or prevention, the method comprising administering to the subject a liposomal α polyglutamated methotrexate composition of any one of [13] to [69 ];
[74] A method of killing a hyperproliferative cell, said method comprising contacting a hyperproliferative cell with a composition as described in any of [1] to [69 ];
[75] a method of killing hyperproliferative cells, comprising contacting hyperproliferative cells with a liposomal α polyglutamated methotrexate composition of any one of [13] to [69 ];
[76] the method of [74] or [75], wherein the hyperproliferative cell is a cancer cell, a mammalian cell, and/or a human cell;
[77] a method for treating cancer, the method comprising administering to a subject having or at risk of having cancer an effective amount of the composition of any one of [1] to [69 ];
[78] a method for treating cancer, the method comprising administering to a subject having or at risk of having cancer an effective amount of the liposomal α polyglutamated methotrexate composition of any one of [13] to [68 ];
[79] the method of [77] or [78], wherein the cancer is selected from the group consisting of: non-hematologic malignancies including, for example, lung cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer, bile duct cancer, gall bladder cancer, sarcomas (e.g., osteosarcoma), brain cancer, central nervous system cancer, and melanoma; and hematological malignancies such as leukemia, lymphoma and other B cell malignancies, myeloma and other plasma cell dyscrasias;
[80] The method of [77] or [78], wherein the cancer is a member selected from the group consisting of: lung, breast, colon, pancreatic, gastric, bladder, head and neck, ovarian and cervical cancer;
[81] the method of [77] or [78], wherein the cancer is mesothelioma or non-small cell lung cancer (NSCLC);
[82] the method of [77] or [78], wherein the cancer is selected from the group consisting of: colorectal cancer, breast cancer, ovarian cancer, lung cancer, head and neck cancer, pancreatic cancer, gastric cancer, and mesothelioma;
[83] a method for treating cancer, the method comprising administering to a subject having or at risk of having cancer cells expressing on the surface a folate receptor bound by a targeting moiety an effective amount of the Lp- α PMTX composition of any one of [50] - [66 ].
[84] A maintenance therapy for a subject undergoing or having undergone cancer therapy, the maintenance therapy comprising administering to the subject undergoing or having undergone cancer therapy an effective amount of the composition of any one of [1] - [69 ];
[85] a maintenance therapy comprising administering to a subject undergoing or having undergone cancer therapy an effective amount of the liposomal α polyglutamated methotrexate composition of any one of [13] to [69 ];
[86] A method for treating an immune system disorder, the method comprising administering to a subject having or at risk of having an immune system disorder an effective amount of the composition of any one of [1] to [69 ];
[87] a method for treating an immune system disorder, the method comprising administering to a subject having or at risk of having an immune system disorder an effective amount of the liposomal α -polyglutamated methotrexate composition of any one of [8] to [69 ];
[88] a method for treating the following diseases:
(a) an infectious disease, the method comprising administering to a subject having or at risk of having an infectious disease an effective amount of the composition according to any one of [1] to [69 ];
(b) an infectious disease, a cardiovascular disease, a metabolic disease, or another disease, the method comprising administering to a subject having or at risk of having an infectious disease, a cardiovascular disease, or another disease an effective amount of a composition according to any one of [1] - [69], wherein the disease is a member selected from: atherosclerosis, cardiovascular disease (CVD), coronary artery disease, myocardial infarction, stroke, metabolic syndrome, gestational trophoblastic disease, and ectopic pregnancy;
(c) An autoimmune disease, the method comprising administering to a subject having or at risk of having an autoimmune disease an effective amount of a composition according to any one of [1] to [69 ];
(d) rheumatoid arthritis, the method comprising administering to a subject having or at risk of having rheumatoid arthritis an effective amount of the composition according to any one of [1] to [69 ];
(e) an inflammatory disorder, the method comprising administering to a subject having or at risk of having inflammation an effective amount of a composition according to any one of [1] - [69], optionally wherein the inflammation is acute, chronic and/or systemic inflammation; or
(f) A skin condition, the method comprising administering to a subject having or at risk of having a skin condition an effective amount of the composition according to any one of [1] - [69], optionally wherein the skin condition is psoriasis;
[89] a method for treating an infectious disease, the method comprising administering to a subject having or at risk of having an infectious disease an effective amount of the liposomal α polyglutamated methotrexate composition of any one of [13] to [69 ];
[90] A method of delivering alpha polyglutamated methotrexate to a tumor that expresses a folate receptor on the surface, the method comprising: administering to a subject having the tumor an amount of an Lp-alpha PMTX composition of any one of [1] - [69] to deliver a therapeutically effective dose of alpha polyglutamated methotrexate to the tumor;
[91] a method of preparing an alpha polyglutamated methotrexate composition comprising a liposomal alpha polyglutamated methotrexate composition of any one of [13] to [69], the method comprising: forming a mixture comprising a liposome component and an alpha polyglutamated antifolate agent in solution; homogenizing the mixture to form liposomes in the solution; and processing the mixture to form liposomes containing alpha-polyglutamated methotrexate;
[92] a method of making the composition of any one of [13] to [69], the method comprising the steps of: forming a mixture comprising a liposome component and alpha-polyglutamated methotrexate in a solution; homogenizing the mixture to form liposomes in the solution; processing the mixture to form liposomes encapsulating and/or encapsulating alpha polyglutamated methotrexate; and providing a targeting moiety on the surface of the liposome, the targeting moiety having specific affinity for at least one of folate receptor alpha (FR-a), folate receptor beta (FR- β), and folate receptor (FR-);
[93] The method of [92], wherein the processing step comprises one or more of: film hydration, extrusion, online mixing, an ethanol injection technology, a freeze thawing technology, reversed phase evaporation, dynamic high-pressure micro-jet, micro-jet mixing, a multiple emulsion method, a freeze drying multiple emulsion method, 3D printing, a membrane contactor method and stirring;
[94] a method of making the composition of any one of [50] to [69], the method comprising the steps of: forming a mixture comprising a liposome component and alpha-polyglutamated methotrexate in a solution; homogenizing the mixture to form liposomes in the solution; processing the mixture to form liposomes encapsulating and/or encapsulating alpha polyglutamated methotrexate; and providing a targeting moiety on the surface of the liposome, the targeting moiety having specific affinity for at least one of folate receptor alpha (FR-a), folate receptor beta (FR- β), and folate receptor (FR-);
[95] a method of making the composition of any one of [50] to [69], the method comprising the steps of: forming a mixture comprising a liposome component and alpha-polyglutamated methotrexate in a solution; processing the mixture to form liposomes encapsulating and/or encapsulating alpha polyglutamated methotrexate; and providing a targeting moiety on the surface of the liposome, the targeting moiety having specific affinity for at least one of folate receptor alpha (FR-a), folate receptor beta (FR- β), and folate receptor (FR-);
[96] The method of [95], wherein the processing step comprises homogenizing the mixture to form liposomes in the solution;
[97] the method of [92], wherein the processing step comprises one or more of: film hydration, extrusion, online mixing, an ethanol injection technology, a freeze thawing technology, reversed phase evaporation, dynamic high-pressure micro-jet, micro-jet mixing, a multiple emulsion method, a freeze drying multiple emulsion method, 3D printing, a membrane contactor method and stirring; and/or
[98] The method of any of [95] to [97], wherein the processing step comprises one or more steps of altering the size of the liposomes by one or more of extrusion, high pressure microfluidization, and/or sonication steps; and/or
[99] The method of any of [91] to [98], wherein at least 1% of a starting material of alpha-polyglutamated methotrexate is encapsulated or embedded in the liposomes;
alpha polyglutamated methotrexate (alpha PMTX)
The present disclosure generally relates to alpha polyglutamated methotrexate (alpha PMTX) compositions. The alpha PMTX composition comprises at least one glutamyl group having an alpha linkage. These compositions are structurally distinct from the L- γ polyglutamated form of methotrexate (L α PMTX), which is produced by the enzyme folate poly- γ -glutamate synthase (FPGS) in cells during methotrexate treatment.
In some embodiments, the alpha PMTX composition contains 2-20, 2-15, 2-10, 2-5, 2-6, or more than 5 glutamyl groups (including glutamyl groups in methotrexate). In some embodiments, each glutamyl group in the α PMTX has an α linkage in addition to the glutamyl group of methotrexate. In some embodiments, each glutamyl group in the alpha PMTX has an alpha linkage in addition to one or more C-terminal glutamyl groups and a glutamyl group of methotrexate. In some embodiments, each glutamyl group in the α PMTX has an α linkage in addition to one or more C-terminal glutamyl groups. In some embodiments, 2 or more glutamyl groups in the alpha PMTX have a gamma linkage. In some embodiments, at least one glutamyl group of the α -polyglutamated methotrexate has both an α carboxyl linkage and a γ carboxyl linkage. In some embodiments, each glutamyl group in the alpha PMTX is in the L form. In some embodiments, each glutamyl group in the alpha PMTX is in the D form in addition to the glutamyl group of methotrexate. In some embodiments, the alpha PMTX comprises two or more glutamyl groups in L form and one or more glutamyl groups in D form. In some embodiments, the polyglutamate chain of the alpha PMTX is linear (unbranched). In some embodiments, the polyglutamate chain of the alpha PMTX is branched.
In some embodiments, the alpha polyglutamated methotrexate is diglutamated. That is, the α -polyglutamated methotrexate contains 1 additional glutamyl group (α MTX-PG) in addition to the glutamyl group of methotrexate1) And the additional glutamyl group is linked to a glutamyl group in the methotrexate by an alpha linkage. In some embodiments, each glutamyl group of α -diglutated methotrexate is in the L form. In other embodiments, the α -di-glutamated MTX comprises glutamyl in the D form.
In some embodiments, the alpha polyglutamated methotrexate is triglutamated. That is, the α -polyglutamated methotrexate contains 2 additional glutamyl groups (α MTX-PG) in addition to the glutamyl groups of methotrexate2). In some embodiments, each of the 2 additional glutamyl groups has an alpha linkage. In other embodiments, one of the 2 additional glutamyl groups has an alpha linkage and the other glutamyl group has a gamma linkage. In some embodiments, one of the 2 additional glutamyl groups has an alpha linkage. In some embodiments, one of the 2 additional glutamyl groups has a gamma linkage. In some embodiments, two of the three glutamyl groups have an alpha linkage. In other embodiments, one of the three glutamyl groups has an alpha linkage and the other glutamyl group has a gamma linkage. In some embodiments, one glutamyl group has both an alpha linkage and a gamma linkage. In some embodiments, each glutamyl group of α tri-glutamated methotrexate is in the L form. In other embodiments, the α tri-glutamated MTX comprises glutamyl in the D form. In other embodiments, each glutamyl group of the α triglutamated methotrexate is in the D form in addition to the glutamyl group of methotrexate. In further embodiments, the tri-glutamated MTX comprises a glutamyl group in the D form and two or more glutamyl groups in the L form. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chains are branched.
In some embodiments, the alpha polyglutamated methotrexate is tetraglutamated, and thus contains 3 additional glutamyl groups in addition to the glutamyl groups in methotrexate (alpha MTX-PG)3). In some embodiments, each of the 3 additional glutamyl groups has an alpha linkage. In other embodiments, 1 or 2 of the 3 additional glutamyl groups have an alpha linkage and the remaining 2 or 1 glutamyl groups have a gamma linkage, respectively. In some embodiments, 2 of the 3 additional glutamyl groups have an α 0 linkage. In other embodiments, one of the 3 additional glutamyl groups has an alpha linkage and the other additional glutamyl group has a gamma linkage. In other embodiments, one of the 3 additional glutamyl groups has both an alpha linkage and a gamma linkage. In other embodiments, three of the four glutamyl groups have an alpha linkage. In some embodiments, at least one glutamyl group has both an alpha linkage and a gamma linkage. In some embodiments, the α tetraglutamated MTX comprises two or more glutamyl groups in the L form. In other embodiments, each glutamyl group of the α -tetraglutamated methotrexate is in the L form. In other embodiments, the α tetraglutamated MTX comprises glutamyl in the D form. In other embodiments, each glutamyl group of the α -tetraglutamated methotrexate is in the D form in addition to a glutamyl group of methotrexate. In further embodiments, the tetraglutamated MTX comprises a glutamyl group in the D form and two or more glutamyl groups in the L form. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chains are branched.
In some embodiments, the alpha polyglutamated methotrexate is pentaglutamated (alpha MTX-PG)4) And contains a chain of 4 additional glutamyl groups linked to the glutamyl group of methotrexate. In some embodiments, each of the 4 additional glutamyl groups in the chain has an alpha linkage. In some embodiments, the amino acid sequence is selected from the group consisting of amino acids, aminoEach of the 4 additional glutamyl groups in the chain has an alpha linkage. In other embodiments, 1, 2, or 3 of the 4 additional glutamyl groups have an alpha linkage and the remaining 3, 2, or 1 glutamyl groups are each linked to a glutamyl group of the molecule by a gamma linkage. In other embodiments, 1 or 2 of the 4 additional glutamyl groups have an alpha linkage and the remaining non-C-terminal glutamyl groups are connected to the glutamyl group of the molecule by a gamma linkage. In some embodiments, at least one additional glutamyl group has both an alpha linkage and a gamma linkage. In some embodiments, at least one of the 5 glutamyl groups has both an alpha linkage and a gamma linkage. In some embodiments, each of the 5 glutamyl groups in the chain has an alpha linkage in addition to the one or more C-terminal glutamyl groups. In some embodiments, the α pentaglutamated MTX comprises two or more glutamyl groups in the L form. In other embodiments, each glutamyl group of the α pentaglutaminated methotrexate is in the L form. In other embodiments, the α pentaglutamated MTX comprises glutamyl in the D form. In other embodiments, each glutamyl group of the α pentaglutaminated methotrexate is in the D form in addition to the glutamyl group of methotrexate. In further embodiments, the pentaglutamated MTX comprises a glutamyl group in the D form and two or more glutamyl groups in the L form. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chains are branched.
In some embodiments, the alpha polyglutamated methotrexate is hexaglutamated (alpha MTX-PG)5) And contains a chain of 5 additional glutamyl groups linked to the glutamyl group of methotrexate. In some embodiments, each of the 5 additional glutamyl groups in the chain has an alpha linkage. In some embodiments, each of the 5 additional glutamyl groups in the chain have an alpha linkage in addition to the one or more C-terminal glutamyl groups. In some embodiments, 4 of the 5 additional glutamyl groups in the chain have an alpha linkage. In other embodimentsIn one embodiment, 1, 2, 3 or 4 of the 5 additional glutamyl groups are connected to the glutamyl group of the molecule by an alpha linkage, and the remaining 4, 3, 2 or 1 glutamyl groups are connected to the glutamyl group of the molecule by a gamma linkage, respectively. In other embodiments, 1, 2, 3, or 4 of the 5 additional glutamyl groups have an alpha linkage, and the remaining non-C-terminal glutamyl groups are linked to the glutamyl groups of the molecule by gamma linkages. In some embodiments, at least one additional glutamyl group has both an alpha linkage and a gamma linkage. In some embodiments, at least one of the 6 glutamyl groups has both an alpha linkage and a gamma linkage. In some embodiments, each of the 6 glutamyl groups, except for one or more C-terminal glutamyl groups, has an alpha linkage. In some embodiments, 5 of the 6 glutamyl groups have an alpha linkage. In some embodiments, the α hexaglutamated MTX comprises two or more glutamyl groups in the L form. In other embodiments, each glutamyl group of the α -hexaglutaminated methotrexate is in the L form. In other embodiments, the α hexaglutamated MTX comprises glutamyl in the D form. In other embodiments, each glutamyl group of the α -hexaglutaminated methotrexate is in the D form in addition to the glutamyl group of methotrexate. In further embodiments, the hexaglutamated MTX comprises a glutamyl group in the D form and two or more glutamyl groups in the L form. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chains are branched.
In some embodiments, the alpha polyglutamated methotrexate is heptaglutamated (alpha MTX-PG)6) And thus contains a chain of 6 additional glutamyl groups linked to the glutamyl groups of methotrexate. In some embodiments, each of the 6 additional glutamyl groups has an alpha linkage. In some embodiments, each of the 6 additional glutamyl groups in the chain have an alpha linkage in addition to the one or more C-terminal glutamyl groups. In some embodiments, 5 of the 6 additional glutamyl groups in the chain areWith an alpha linkage. In other embodiments, 1, 2, 3, 4, or 5 of the 6 additional glutamyl groups have an alpha linkage and the remaining 5, 4, 3, 2, or 1 glutamyl groups each have a gamma linkage. In other embodiments, 1, 2, 3, 4, or 5 of the 6 additional glutamyl groups have an α 0 linkage, and the remaining non-C-terminal glutamyl groups are connected to the glutamyl groups of the molecule by γ linkages. In some embodiments, at least one additional glutamyl group has both an alpha linkage and a gamma linkage. In some embodiments, at least one of the 7 glutamyl groups has both an alpha linkage and a gamma linkage. In some embodiments, each of the 7 glutamyl groups has an alpha linkage in addition to one or more C-terminal glutamyl groups. In some embodiments, 6 of the 7 glutamyl groups have an alpha linkage. In some embodiments, the alpha heptaglutamated MTX comprises two or more glutamyl groups in the L form. In other embodiments, each glutamyl group of the α heptaglutamated methotrexate is in the L form. In other embodiments, the alpha heptaglutamated MTX comprises glutamyl in the D form. In other embodiments, each glutamyl group of the α heptaglutamated methotrexate is in the D form in addition to a glutamyl group of methotrexate. In further embodiments, the heptaglutamated MTX comprises a glutamyl group in the D form and two or more glutamyl groups in the L form. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chains are branched.
In some embodiments, the alpha polyglutamated methotrexate is octaglutamated (alpha MTX-PG)7) And thus contains a chain of 7 additional glutamyl groups linked to the glutamyl groups of methotrexate. In some embodiments, each of the 7 additional glutamyl groups in the chain have an alpha linkage in addition to the one or more C-terminal glutamyl groups. In some embodiments, 6 of the 7 additional glutamyl groups in the chain have an alpha linkage. In some embodiments, each of the 7 additional glutamyl groups hasThere is an alpha linkage. In other embodiments, 1, 2, 3, 4, 5, or 6 of the 7 additional glutamyl groups have an alpha linkage and the remaining 6, 5, 4, 3, 2, or 1 glutamyl groups have gamma linkages, respectively. In other embodiments, 1, 2, 3, 4, 5, or 6 of the 7 additional glutamyl groups have an α 0 linkage, and the remaining non-C-terminal glutamyl groups are connected to the glutamyl groups of the molecule by γ linkages. In some embodiments, at least one additional glutamyl group has both an alpha linkage and a gamma linkage. In some embodiments, at least one of the 8 glutamyl groups has both an alpha linkage and a gamma linkage. In some embodiments, each of the 8 glutamyl groups has an alpha linkage in addition to one or more C-terminal glutamyl groups. In some embodiments, 7 of the 8 glutamyl groups have an alpha linkage. In some embodiments, the α octaglutamated MTX comprises two or more glutamyl groups in the L form. In other embodiments, each glutamyl group of the α octaglutamatemethotrexate is in the L form. In other embodiments, the α octaglutamated MTX comprises glutamyl in the D form. In other embodiments, each glutamyl group of the α octaglutamated methotrexate is in the D form in addition to a glutamyl group of methotrexate. In further embodiments, the octaglutamated MTX comprises a glutamyl group in the D form and two or more glutamyl groups in the L form. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chains are branched.
In some embodiments, the alpha polyglutamated methotrexate is nonaglutamated (alpha MTX-PG)8) And contains a chain of 8 additional glutamyl groups linked to the glutamyl group of methotrexate. In some embodiments, each of the 8 additional glutamyl groups in the chain have an alpha linkage in addition to the one or more C-terminal glutamyl groups. In some embodiments, 7 of the 8 additional glutamyl groups in the chain have an alpha linkage. In some embodiments, each of the 8 additional glutamyl groupsOne each having an alpha linkage. In other embodiments, 1, 2, 3, 4, 5, 6, or 7 of the 8 additional glutamyl groups have an alpha linkage and the remaining 7, 6, 5, 4, 3, 2, or 1 glutamyl groups have a gamma linkage, respectively. In other embodiments, 1, 2, 3, 4, 5, 6, or 7 of the 8 additional glutamyl groups have an α 0 linkage, and the remaining non-C-terminal glutamyl groups are connected to the glutamyl groups of the molecule by γ linkages. In some embodiments, at least one additional glutamyl group has both an alpha linkage and a gamma linkage. In some embodiments, at least one of the 9 glutamyl groups has both an alpha linkage and a gamma linkage. In some embodiments, each of the 9 glutamyl groups, except for one or more C-terminal glutamyl groups, has an alpha linkage. In some embodiments, 8 of the 9 glutamyl groups have an alpha linkage. In some embodiments, the α nonaglutamated MTX comprises two or more glutamyl groups in the L form. In other embodiments, each glutamyl group of the α nonaglutamated methotrexate is in the L form. In other embodiments, the α nonaglutamated MTX comprises glutamyl in the D form. In other embodiments, each glutamyl group of the α nonaglutamated methotrexate is in the D form in addition to a glutamyl group of methotrexate. In further embodiments, the nonaglutamated MTX comprises a glutamyl group in the D form and two or more glutamyl groups in the L form. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chains are branched.
In some embodiments, the alpha polyglutamated methotrexate is decaglutamated (alpha MTX-PG)9) (i.e., a chain containing 9 additional glutamyl groups linked to the glutamyl groups of methotrexate). In some embodiments, each of the 9 additional glutamyl groups has an alpha linkage. In some embodiments, each of the 9 additional glutamyl groups in the chain have an alpha linkage in addition to the one or more C-terminal glutamyl groups. In some embodiments, the chain8 of the 9 additional glutamyl groups have an alpha linkage. In other embodiments, 1, 2, 3, 4, 5, 6, 7, or 8 of the 9 additional glutamyl groups have an alpha linkage and the remaining 8, 7, 6, 5, 4, 3, 2, or 1 glutamyl groups have a gamma linkage, respectively. In other embodiments, 1, 2, 3, 4, 5, 6, 7, or 8 of the 9 additional glutamyl groups have an α 0 linkage, and the remaining non-C-terminal glutamyl groups are linked to the glutamyl groups of the molecule by γ linkages. In some embodiments, at least one additional glutamyl group has both an alpha linkage and a gamma linkage. In some embodiments, at least one of the 10 glutamyl groups has both an alpha linkage and a gamma linkage. In some embodiments, each of the 10 glutamyl groups has an alpha linkage in addition to one or more C-terminal glutamyl groups. In some embodiments, 9 of the 10 glutamyl groups have an alpha linkage. In some embodiments, the α decaglutamated MTX comprises two or more glutamyl groups in the L form. In other embodiments, each glutamyl group of the α decaglutamated methotrexate is in the L form. In other embodiments, the α decaglutamated MTX comprises glutamyl in the D form. In other embodiments, each glutamyl group of the α decaglutamated methotrexate is in the D form in addition to a glutamyl group of methotrexate. In further embodiments, the decaglutamated MTX comprises a glutamyl group in the D form and two or more glutamyl groups in the L form. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chains are branched.
In some embodiments, the alpha polyglutamated methotrexate is undeglutamated (alpha MTX-PG)10). In some embodiments, each of the 10 additional glutamyl groups has an alpha linkage. In some embodiments, each of the 10 additional glutamyl groups in the chain have an alpha linkage in addition to the one or more C-terminal glutamyl groups. In some embodiments, 10 additional valleys in the chain9 of the aminoacyl groups have an alpha linkage. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, or 9 of the 10 additional glutamyl groups have an alpha linkage and the remaining 9, 8, 7, 6, 5, 4, 3, 2, or 1 glutamyl groups have gamma linkages, respectively. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, or 9 of the 10 additional glutamyl groups have an α 0 linkage, and the remaining non-C-terminal glutamyl groups are linked to the glutamyl groups of the molecule by γ linkages. In some embodiments, at least one additional glutamyl group has both an alpha linkage and a gamma linkage. In some embodiments, at least one of the 11 glutamyl groups has both an alpha linkage and a gamma linkage. In some embodiments, each of the 11 glutamyl groups has an alpha linkage in addition to one or more C-terminal glutamyl groups. In some embodiments, 10 of the 11 glutamyl groups have an alpha linkage. In some embodiments, the α undeglutamated MTX comprises two or more glutamyl groups in the L form. In other embodiments, each glutamyl group of the α undeglutamatemethotrexate is in the L form. In other embodiments, the α undeglutamated MTX comprises D glutamyl. In other embodiments, each glutamyl group of the α undeglutamated methotrexate is in the D form in addition to a glutamyl group of methotrexate. In further embodiments, the undeglutamated MTX comprises a glutamyl group in the D form and two or more glutamyl groups in the L form. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chains are branched.
In some embodiments, the alpha polyglutamated methotrexate is dodecaglutamated (alpha MTX-PG)11). In some embodiments, each of the 11 additional glutamyl groups has an alpha linkage. In some embodiments, each of the 11 additional glutamyl groups in the chain have an alpha linkage in addition to the one or more C-terminal glutamyl groups. In some embodiments, 11 additional of the chains are present10 of the glutamyl groups of (a) have an alpha linkage. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the 11 additional glutamyl groups have an alpha linkage and the remaining 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 glutamyl groups have gamma linkages, respectively. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the 11 additional glutamyl groups have an alpha 0 linkage, and the remaining non-C-terminal glutamyl groups are linked to the glutamyl groups of the molecule by gamma linkages. In some embodiments, at least one additional glutamyl group has both an alpha linkage and a gamma linkage. In some embodiments, at least one of the 12 glutamyl groups has both an alpha linkage and a gamma linkage. In some embodiments, each of the 12 glutamyl groups has an alpha linkage in addition to one or more C-terminal glutamyl groups. In some embodiments, 11 of the 12 glutamyl groups have an alpha linkage. In some embodiments, the α dodecaglutamated MTX comprises two or more glutamyl groups in the L form. In other embodiments, each glutamyl group of the α -dodecaglutamated methotrexate is in the L form. In other embodiments, the α dodecaglutamated MTX comprises glutamyl in the D form. In other embodiments, each glutamyl group of the α -dodecaglutamated methotrexate is in the D form in addition to the glutamyl group of methotrexate. In further embodiments, the dodecaglutamated MTX comprises glutamyl in the D form and two or more glutamyl in the L form. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chains are branched.
In some embodiments, the alpha polyglutamated methotrexate is tridecyl glutamated (alpha MTX-PG)12). In some embodiments, each of the 12 additional glutamyl groups has an alpha linkage. In some embodiments, each of the 12 additional glutamyl groups in the chain has an alpha linkage in addition to the one or more C-terminal glutamyl groups. In some casesIn embodiments, 11 of the 12 additional glutamyl groups in the chain have an alpha linkage. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 of the 12 additional glutamyl groups have an alpha linkage and the remaining 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 glutamyl group, respectively, have a gamma linkage. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 of the 12 additional glutamyl groups have an alpha 0 linkage, and the remaining non-C-terminal glutamyl groups are linked to the glutamyl groups of the molecule by gamma linkages. In some embodiments, at least one additional glutamyl group has both an alpha linkage and a gamma linkage. In some embodiments, at least one of the 13 glutamyl groups has both an alpha linkage and a gamma linkage. In some embodiments, each of the 13 glutamyl groups has an alpha linkage in addition to one or more C-terminal glutamyl groups. In some embodiments, 12 of the 13 glutamyl groups have an alpha linkage. In some embodiments, the α tridecylated MTX comprises two or more glutamyl groups in the L form. In other embodiments, each glutamyl group of the α tridecyl-glutamated methotrexate is in the L form. In other embodiments, the α tridecylated MTX comprises glutamyl in the D form. In other embodiments, each glutamyl group of the α tridecyl-glutamated methotrexate is in the D form in addition to the glutamyl group of methotrexate. In further embodiments, the tridecylated MTX comprises a glutamyl group in the D form and two or more glutamyl groups in the L form. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chains are branched.
In some embodiments, the alpha polyglutamated methotrexate is tetradecaglutated (alpha MTX-PG)13). In some embodiments, each of the 13 additional glutamyl groups has an alpha linkage. In some embodiments, 13 in the chain is other than one or more C-terminal glutamyl groupsEach of the additional glutamyl groups has an alpha linkage. In some embodiments, 12 of the 13 additional glutamyl groups in the chain have an alpha linkage. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 of the 13 additional glutamyl groups have an alpha 0 linkage and the remaining 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 glutamyl groups have a gamma linkage, respectively. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 of the 13 additional glutamyl groups have an α 1 linkage and the remaining non-C-terminal glutamyl groups are linked to the glutamyl groups of the molecule by a γ linkage. In some embodiments, at least one additional glutamyl group has both an alpha linkage and a gamma linkage. In some embodiments, at least one of the 14 glutamyl groups has both an alpha linkage and a gamma linkage. In some embodiments, each of the 14 glutamyl groups has an alpha linkage in addition to one or more C-terminal glutamyl groups. In some embodiments, 13 of the 14 glutamyl groups have an alpha linkage. In some embodiments, the α tetradecanoized MTX comprises two or more glutamyl groups in the L form. In other embodiments, each glutamyl group of the α tetradecahlutamate methotrexate is in the L form. In other embodiments, the α tetradecanoized MTX comprises glutamyl in the D form. In other embodiments, each glutamyl group of the α -tetradecylglutamated methotrexate is in the D form in addition to a glutamyl group of methotrexate. In further embodiments, the tetradecanoized MTX comprises a glutamyl group in the D form and two or more glutamyl groups in the L form. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chains are branched.
In some embodiments, the alpha polyglutamated methotrexate is pentadecylated (alpha MTX-PG)14). In some embodiments, each of the 14 additional glutamyl groups has an alpha linkage. In some embodiments, each of the 14 additional glutamyl groups in the chain has an alpha linkage in addition to the one or more C-terminal glutamyl groups. In some embodiments, 13 of the 14 additional glutamyl groups in the chain have an alpha linkage. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 of the 14 additional glutamyl groups have an alpha 0 linkage and the remaining 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 glutamyl group, respectively, have a gamma linkage. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 of the 14 additional glutamyl groups have an α 1 linkage and the remaining non-C-terminal glutamyl groups are linked to the glutamyl group of the molecule by a γ linkage. In some embodiments, at least one additional glutamyl group has both an alpha linkage and a gamma linkage. In some embodiments, at least one of the 15 glutamyl groups has both an alpha linkage and a gamma linkage. In some embodiments, each of the 15 glutamyl groups has an alpha linkage in addition to one or more C-terminal glutamyl groups. In some embodiments, 14 of the 15 glutamyl groups have an alpha linkage. In some embodiments, the α pentadecglutamated MTX comprises two or more glutamyl groups in the L form. In other embodiments, each glutamyl group of the α pentadecyl methotrexate is in the L form. In other embodiments, the α pentadecglutamated MTX comprises glutamyl in the D form. In other embodiments, each glutamyl group of the α pentadecyl-glutamated methotrexate is in the D form in addition to a glutamyl group of methotrexate. In further embodiments, the pentadecylated MTX comprises glutamyl in the D form and two or more glutamyl in the L form. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chains are branched.
In some embodiments, the alpha-polyglutamated methotrexate is cetylglutamated (c)αMTX-PG15). In some embodiments, each of the 15 additional glutamyl groups has an alpha linkage. In some embodiments, each of the 15 additional glutamyl groups in the chain have an alpha linkage in addition to the one or more C-terminal glutamyl groups. In some embodiments, 14 of the 15 additional glutamyl groups in the chain have an α 0 linkage. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 of the 15 additional glutamyl groups have an α 1 linkage and the remaining 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 glutamyl groups have a γ linkage, respectively. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 of the 15 additional glutamyl groups have an α 2 linkage and the remaining non-C-terminal glutamyl groups are linked to the glutamyl groups of the molecule by a γ linkage. In some embodiments, at least one additional glutamyl group has both an alpha linkage and a gamma linkage. In some embodiments, at least one of the 16 glutamyl groups has both an alpha linkage and a gamma linkage. In some embodiments, each of the 16 glutamyl groups has an alpha linkage in addition to one or more C-terminal glutamyl groups. In some embodiments, 15 of the 16 glutamyl groups have an alpha linkage. In some embodiments, the α hexadecagoylated MTX comprises two or more glutamyl groups in the L form. In other embodiments, each glutamyl group of the α -hexadecagonomic methotrexate is in the L form. In other embodiments, the α hexadecimated MTX comprises glutamyl in the D form. In other embodiments, each glutamyl group of the α -hexadecagoylated methotrexate is in the D form in addition to the glutamyl group of methotrexate. In further embodiments, the cetylglutamated MTX comprises a glutamyl group in the D form and two or more glutamyl groups in the L form. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chain Is branched.
In other embodiments, the alpha polyglutamated methotrexate is heptadecaglutamated (alpha MTX-PG)16). In some embodiments, each of the 16 additional glutamyl groups has an alpha linkage. In some embodiments, each of the 16 additional glutamyl groups in the chain has an alpha linkage in addition to the one or more C-terminal glutamyl groups. In some embodiments, 15 of the 16 additional glutamyl groups in the chain have an α 0 linkage. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 of the 16 additional glutamyl groups have an α 1 linkage and the remaining 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 glutamyl group has a γ linkage, respectively. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 of the 16 additional glutamyl groups have an α 2 linkage and the remaining non-C-terminal glutamyl groups are linked to the glutamyl group of the molecule by a γ linkage. In some embodiments, at least one additional glutamyl group has both an alpha linkage and a gamma linkage. In some embodiments, at least one of the 17 glutamyl groups has both an alpha linkage and a gamma linkage. In some embodiments, each of the 17 glutamyl groups has an alpha linkage in addition to one or more C-terminal glutamyl groups. In some embodiments, 16 of the 17 glutamyl groups have an alpha linkage. In some embodiments, the α heptadecaglutamic MTX comprises two or more glutamyl groups in the L form. In other embodiments, each glutamyl group of the α heptadecaglutamic methotrexate is in the L form. In other embodiments, the α heptadecaglutamic MTX comprises D glutamyl. In other embodiments, each glutamyl group of the α heptadecaglutamated methotrexate is in the D form in addition to a glutamyl group of methotrexate. In a further embodiment, the heptadecaglutamated MTX comprises glutamyl in the D form and two or more A plurality of glutamyl groups in the form of L. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chains are branched.
In some embodiments, the alpha polyglutamated methotrexate is octadecylglutamated (alpha MTX-PG)17). In some embodiments, each of the 17 additional glutamyl groups has an alpha linkage. In some embodiments, each of the 17 additional glutamyl groups in the chain have an alpha linkage in addition to the one or more C-terminal glutamyl groups. In some embodiments, 16 of the 17 additional glutamyl groups in the chain have an α 0 linkage. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 of the 17 additional glutamyl groups have an α 1 linkage and the remaining 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 glutamyl groups have γ linkages, respectively. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 of the 17 additional glutamyl groups have an alpha linkage and the remaining non-C-terminal glutamyl groups are linked to the glutamyl group of the molecule by a gamma linkage. In some embodiments, at least one additional glutamyl group has both an alpha linkage and a gamma linkage. In some embodiments, at least one of the 18 glutamyl groups has both an alpha linkage and a gamma linkage. In some embodiments, each of the 18 glutamyl groups has an alpha linkage in addition to one or more C-terminal glutamyl groups. In some embodiments, 17 of the 18 glutamyl groups have an alpha linkage. In some embodiments, the α octadecylated MTX comprises two or more glutamyl groups in the L form. In other embodiments, each glutamyl group of the α octadecylglutamated methotrexate is in the L form. In other embodiments, the α octadecylated MTX comprises glutamyl in the D form. In other embodiments, glutamyl other than methotrexate Additionally, each glutamyl group of the α -octadecylglutamated methotrexate is in the D form. In further embodiments, the octadecylated MTX comprises glutamyl in the D form and two or more glutamyl in the L form. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chains are branched.
In some embodiments, the alpha polyglutamated methotrexate is nonadecaglutamated (alpha MTX-PG)18). In some embodiments, each of the 18 additional glutamyl groups has an alpha linkage. In some embodiments, each of the 18 additional glutamyl groups in the chain has an alpha linkage in addition to the one or more C-terminal glutamyl groups. In some embodiments, 17 of the 18 additional glutamyl groups in the chain have an alpha linkage. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 of the 18 additional glutamyl groups have an alpha linkage and the remaining 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 glutamyl groups have gamma linkages, respectively. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 of the 18 additional glutamyl groups have an alpha linkage and the remaining non-C-terminal glutamyl groups are linked to the glutamyl groups of the molecule by gamma linkages. In some embodiments, at least one additional glutamyl group has both an alpha linkage and a gamma linkage. In some embodiments, at least one of the 19 glutamyl groups has both an alpha linkage and a gamma linkage. In some embodiments, each of the 19 glutamyl groups has an alpha linkage in addition to one or more C-terminal glutamyl groups. In some embodiments, 18 of the 19 glutamyl groups have an alpha linkage. In some embodiments, the α nineteen glutamated MTX comprises two or more glutamyl groups in the L form. In other embodiments In this embodiment, each glutamyl group of the α nineteen glutamated methotrexate is in the L form. In other embodiments, the α nineteen glutamated MTX comprises D glutamyl. In other embodiments, each glutamyl group of the α nonadecaglutamated methotrexate is in the D form in addition to a glutamyl group of methotrexate. In further embodiments, the nineteen glutamated MTX comprises a glutamyl group in the D form and two or more glutamyl groups in the L form. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chains are branched.
In some embodiments, the alpha polyglutamated methotrexate is icosiglitated (alpha MTX-PG)19). In some embodiments, each of the 19 additional glutamyl groups has an alpha linkage. In some embodiments, each of the 19 additional glutamyl groups in the chain has an alpha linkage in addition to the one or more C-terminal glutamyl groups. In some embodiments, 18 of the 19 additional glutamyl groups in the chain have an alpha linkage. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 of the 19 additional glutamyl groups have an alpha linkage and the remaining 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 glutamyl group respectively have a gamma linkage. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 of the 19 additional glutamyl groups have an alpha linkage and the remaining non-C-terminal glutamyl groups are linked to the glutamyl group of the molecule by a gamma linkage. In some embodiments, at least one additional glutamyl group has both an alpha linkage and a gamma linkage. In some embodiments, at least one of the 20 glutamyl groups has both an alpha linkage and a gamma linkage. In some embodiments, the 20 glutamyl groups are in addition to one or more C-terminal glutamyl groups Each of the radicals has an alpha linkage. In some embodiments, 19 of the 20 glutamyl groups have an alpha linkage. In some embodiments, the alpha eicosylglutamated MTX comprises two or more glutamyl groups in the L form. In other embodiments, each glutamyl group of the α -eicosatetrapterin is in the L form. In other embodiments, the alpha eicosylglutamated MTX comprises glutamyl in the D form. In other embodiments, each glutamyl group of the α -eicosatetraopterin is in the D form in addition to the glutamyl group of methotrexate. In further embodiments, the icosaglutamated MTX comprises a glutamyl group in the D form and two or more glutamyl groups in the L form. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chains are branched.
In some embodiments, the alpha polyglutamated methotrexate is icosoglutamated (alpha MTX-PG)20). In some embodiments, each of the 20 additional glutamyl groups has an alpha linkage. In some embodiments, each of the 20 additional glutamyl groups in the chain has an alpha linkage in addition to the one or more C-terminal glutamyl groups. In some embodiments, 19 of the 20 additional glutamyl groups in the chain have an alpha linkage. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 of the 20 additional glutamyl groups have an alpha linkage and the remaining 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 glutamyl group has a gamma linkage, respectively. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 of the 20 additional glutamyl groups have an alpha linkage, and the remaining non-C-terminal glutamyl groups are linked to the glutamyl group of the molecule by a gamma linkage And (4) a base. In some embodiments, at least one additional glutamyl group has both an alpha linkage and a gamma linkage. In some embodiments, at least one of the 21 glutamyl groups has both an alpha linkage and a gamma linkage. In some embodiments, each of the 21 glutamyl groups has an alpha linkage in addition to one or more C-terminal glutamyl groups. In some embodiments, 20 of the 21 glutamyl groups have an alpha linkage. In some embodiments, the α heneicosylated MTX comprises two or more glutamyl groups in the L form. In other embodiments, each glutamyl group of the α heneico-glutamated methotrexate is in the L form. In other embodiments, the α heneicosylated MTX comprises glutamyl in the D form. In other embodiments, each glutamyl group of the α -heneico-glutamated methotrexate is in the D form in addition to the glutamyl group of methotrexate. In further embodiments, the heneicosyl MTX comprises a glutamyl group in the D form and two or more glutamyl groups in the L form. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chains are branched.
In some embodiments, the α polyglutamated methotrexate contains a chain of 4-7 glutamyl groups linked to methotrexate (i.e., α MTX-PGn, where n ═ 4-7), and each of the 4-7 linked glutamyl groups has an α linkage. In some embodiments, the α polyglutamated methotrexate contains a chain of 4-7 glutamyl groups linked to methotrexate (i.e., α MTX-PGn, where n ═ 4-7), and each of the 4-7 linked glutamyl groups, except for one or more C-terminal glutamyl groups, has an α linkage. In some embodiments, each of the 4-7 linked glutamyl groups is in the L form. In other embodiments, each of the 4-7 linked glutamyl groups is in the D form. In other embodiments, the 4-7 linked glutamyl groups are in the L form and the D form. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chains are branched.
In one embodiment, the alpha polyglutamated methotrexate is tetraglutamated and each of the 3 glutamyl groups in the polyglutamate chain to which methotrexate is attached contains an alpha linkage. In one embodiment, the α polyglutamated methotrexate is tetraglutamated, and each of the 3 glutamyl groups in the polyglutamate chain to which methotrexate is attached contains an α linkage in addition to one or more C-terminal glutamyl groups. In some embodiments, each of the 4 glutamyl groups is in the L form. In some embodiments, each glutamyl group in the α -tetraglutamated methotrexate is in the D form in addition to the glutamyl group of methotrexate. In other embodiments, at least two glutamyl groups in the α -tetraglutamated methotrexate are in the L form and at least one glutamyl group is in the D form. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chains are branched.
In one embodiment, the alpha polyglutamated methotrexate is pentaglutamated and each of the 4 glutamyl groups in the polyglutamate chain to which methotrexate is attached contains an alpha linkage. In one embodiment, the α polyglutamated methotrexate is pentaglutamated and each of the 4 glutamyl groups in the polyglutamate chain to which methotrexate is attached contains an α linkage in addition to one or more C-terminal glutamyl groups. In some embodiments, each of the 4 glutamyl groups is in the L form. In some embodiments, each glutamyl group in the α pentaglutaminated methotrexate is in the D form in addition to the glutamyl group of methotrexate. In other embodiments, at least two glutamyl groups in the α pentaglutaminated methotrexate are in the L form and at least one glutamyl group is in the D form. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chains are branched.
In one embodiment, the alpha polyglutamated methotrexate is hexaglutamated and each of the 5 glutamyl groups in the polyglutamate chain to which methotrexate is attached contains an alpha linkage. In one embodiment, the α polyglutamated methotrexate is hexaglutamated and each of the 5 glutamyl groups in the polyglutamate chain to which methotrexate is attached contains an α linkage in addition to one or more C-terminal glutamyl groups. In some embodiments, each of the 5 glutamyl groups is in the L form. In some embodiments, each glutamyl group in the alpha-hexaglutaminated methotrexate is in the D form in addition to the glutamyl group of methotrexate. In other embodiments, at least two glutamyl groups in the α -hexaglutaminated methotrexate are in the L form and at least one glutamyl group is in the D form. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chains are branched.
In one embodiment, the alpha polyglutamated methotrexate is heptaglutamated and each of the 6 glutamyl groups in the polyglutamate chain to which methotrexate is attached contains an alpha linkage. In another embodiment, the α polyglutamated methotrexate is heptaglutamated and each of the 6 glutamyl groups in the polyglutamate chain to which methotrexate is attached contains an α linkage in addition to one or more C-terminal glutamyl groups. In some embodiments, each of the 6 glutamyl groups is in the L form. In some embodiments, each glutamyl group in the α heptaglutamated methotrexate is in the D form in addition to a glutamyl group of methotrexate. In other embodiments, at least two glutamyl groups in the α heptaglutamated methotrexate are in the L form and at least one glutamyl group is in the D form. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chains are branched.
In some embodiments, the alpha polyglutamated methotrexate (alpha PMTX) contains a total of 1-15, 1-10, 2-15, 2-10, 3-15, 3-10, 3-6, 3-5, 4-10, 4-7, or 4-6 glutamyl groups, including glutamyl groups in methotrexate, or any range therebetween. In some embodiments, each glutamyl group in the α PMTX has an α linkage in addition to the glutamyl group of methotrexate. In some embodiments, each glutamyl group in the alpha PMTX has an alpha linkage in addition to one or more C-terminal glutamyl groups and a glutamyl group of methotrexate. In some embodiments, each glutamyl group in the α PMTX has an α linkage in addition to one or more C-terminal glutamyl groups. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 glutamyl groups in the alpha PMTX have an alpha linkage. In some implementations, the alpha PMTX includes glutamyl in L and D forms. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 glutamyl groups in the alpha PMTX have an alpha linkage, and 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 glutamyl groups have a gamma linkage or none of the glutamyl groups have a gamma linkage, respectively. In some embodiments, each glutamyl group in the polyglutamate structure of the polyglutamated methotrexate is in the L form. In some embodiments, each glutamyl group in the alpha PMTX is in the D form in addition to the glutamyl group of methotrexate. In one embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 glutamyl groups in the alpha PMTX are in the L form. In another embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 glutamyl groups in the alpha PMTX are in the form of D. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chains are branched.
In some embodiments, the alpha polyglutamated methotrexate (alpha PMTX) contains a total of 2-20, 2-15, 2-10, 2-5 glutamyl groups, including glutamyl groups in methotrexate, or any range therebetween. In some embodiments, each glutamyl group in the α PMTX has an α linkage in addition to the glutamyl group of methotrexate. In some embodiments, each glutamyl group in the alpha PMTX has an alpha linkage in addition to one or more C-terminal glutamyl groups and a glutamyl group of methotrexate. In some embodiments, each glutamyl group in the α PMTX has an α linkage in addition to one or more C-terminal glutamyl groups. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 glutamyl groups have an alpha linkage. In some embodiments, the alpha PMTX contains two or more glutamyl groups with gamma linkages. In other embodiments, in addition to the glutamyl groups of methotrexate, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 glutamyl groups in the alpha PMTX have an alpha linkage, and 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 glutamyl groups have a gamma linkage, respectively, or none of the glutamyl groups have a gamma linkage. In some embodiments, each glutamyl group in the alpha PMTX is in the L form. In some embodiments, each glutamyl group in the alpha PMTX is in the D form in addition to the glutamyl group of methotrexate. In one embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 glutamyl groups in the alpha PMTX are in the L form. In another embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 glutamyl groups in the alpha PMTX are in the D form.
In some embodiments, the α -polyglutamated methotrexate contains a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 glutamyl groups in addition to glutamyl groups in methotrexate). In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 additional glutamyl groups have an alpha linkage. In further embodiments, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 glutamyl groups in the alpha polyglutamated methotrexate have a gamma linkage. In some embodiments, at least one glutamyl group has both an alpha linkage and a gamma linkage. In some embodiments, the glutamyl group in methotrexate has an alpha linkage. In some embodiments, the glutamyl group in methotrexate has both an alpha linkage and a gamma linkage.
In some embodiments, a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 glutamyl groups in the alpha polyglutamated methotrexate are in the L form, the D form, or in the L form and the D form. In some embodiments, each glutamyl group of the α -polyglutamated methotrexate is in the L form. In other embodiments, each glutamyl group of the α -polyglutamated methotrexate is in the D form in addition to a glutamyl group of methotrexate. In alternative embodiments, at least two glutamyl groups in the alpha polyglutamated methotrexate are in the L form and at least one glutamyl group in the alpha polyglutamated methotrexate is in the D form. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 glutamyl groups in the alpha polyglutamated methotrexate are in the L form. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 glutamyl groups in the alpha polyglutamated methotrexate are in the D form.
In further embodiments, the alpha polyglutamated methotrexate contains 20-100, 20-75, 20-50, 20-40, 20-30, 20-25, or more than 100 alpha glutamyl groups, or any range therebetween. In some embodiments, each glutamyl group of the α -polyglutamated methotrexate is in the L form. In other embodiments, each glutamyl group of the α -polyglutamated methotrexate is in the D form in addition to a glutamyl group of methotrexate. In alternative embodiments, at least two glutamyl groups in the alpha polyglutamated methotrexate are in the L form and at least one glutamyl group in the alpha polyglutamated methotrexate is in the D form.
In further embodiments, provided compositions comprise alpha polyglutamated methotrexate containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 1-10, or 1-20 glutamyl groups with alpha linkages. In some embodiments, the alpha polyglutamated methotrexate contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 1-10, or 1-20 glutamyl groups in the L form. In some embodiments, the alpha polyglutamated methotrexate contains 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 1-10, or 1-20 glutamyl groups in D form. In some embodiments, the α -polyglutamated methotrexate contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 1-10, or 1-20 glutamyl groups in the L form and 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 1-10 or 1-20 glutamyl groups in the D form. In other embodiments, the alpha polyglutamated methotrexate contains at least 1 glutamyl group having both an alpha linkage and a gamma linkage. In some embodiments, the alpha polyglutamated methotrexate contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 1-10, or more than 10 glutamyl groups having both alpha and gamma linkages.
In some embodiments, the alpha polyglutamated methotrexate contains at least 1 glutamyl group with an alpha linkage, and contains 2, 3, 4, 5, 6, 7, 8, 9, 1-10, 1-20 or more glutamyl groups with a gamma linkage. For example, in some embodiments, the alpha polyglutamated methotrexate contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 1-10L-alpha glutamyl linkages, and also contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 1-10L-gamma glutamyl linkages. In some other embodiments, the alpha polyglutamated methotrexate contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 1-10L-alpha glutamyl linkages, and also contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 1-10D-gamma glutamyl linkages. In still other embodiments, the alpha polyglutamated methotrexate contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 1-10D-alpha glutamyl linkages, and also contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 1-10D-gamma glutamyl linkages. In other embodiments, the alpha-polyglutamated methotrexate contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 1-10D-gamma glutamyl linkages, and also contains 1, 2, 3, 4, 5, 6, or 1-10L-gamma glutamyl linkages. In other embodiments, the alpha polyglutamated methotrexate contains at least 1 glutamyl group having both an alpha linkage and a gamma linkage. In some embodiments, the alpha polyglutamated methotrexate contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 1-10, or more than 10 glutamyl groups having both alpha and gamma linkages.
In some embodiments, the α polyglutamated methotrexate compositions provided herein are capable of accepting one or more additional glutamyl groups, i.e., the compositions are capable of acting as substrates for FPGS (folate polyglutamate synthase). Reagents and assays and reagents for determining the ability of an alpha polyglutamated methotrexate composition to serve as a substrate for FPGS (e.g., human FPGS or rat liver FPGS) are readily available and can be routinely performed.
In some embodiments, the rate of uptake of naked alpha PMTX compositions disclosed herein (e.g., alpha PMTX not associated with a delivery vehicle) by hepatocytes is significantly reduced compared to the rate of uptake of methotrexate under physiological conditions. In some embodiments, the naked α PMTX composition has a hepatocyte uptake rate of less than 30%, 20%, 15%, or 10% compared to the rate of methotrexate. In other embodiments, the rate of efflux (export) of the α PMTX compositions disclosed herein from hepatocytes occurs at a significantly reduced rate (e.g., less than 30%, 20%, 15%, or 10%) compared to the rate of methotrexate.
In some embodiments, the α -polyglutamated methotrexate compositions provided herein are more cytotoxic to hyperproliferative cells than methotrexate. In some embodiments, the hyperproliferative cell is a cancer cell. In some embodiments, the hyperproliferative cell is a colorectal cancer cell, a colon cancer cell, a breast cancer cell, or an ovarian cancer cell. In some embodiments, the cancer cell is a mesothelioma cell or a non-small cell lung cancer cell. In some embodiments, cytotoxicity is measured in an in vitro assay. In some embodiments, the alpha polyglutamated methotrexate is hexaglutamated methotrexate.
In some embodiments, the α -polyglutamated methotrexate compositions provided herein have lower toxic side effects compared to methotrexate. In some embodiments, the α -polyglutamated methotrexate compositions provided herein are less toxic to non-hyperproliferative cells than methotrexate. In some embodiments, the α -polyglutamated methotrexate compositions provided herein are less toxic to neutrophils, hepatocytes, or colonic epithelial cells than methotrexate. In some embodiments, the neutrophil is a human neutrophil, a differentiated human neutrophil, or a neutrophil differentiated from a CD34+ cell. In some embodiments, the hepatocyte is an AML12 hepatocyte. In some embodiments, the colonic epithelial cells are CCD841 colonic epithelial cells. In some embodiments, toxicity is measured in an in vitro assay. In some embodiments, the alpha polyglutamated methotrexate is hexaglutamated methotrexate.
In some embodiments, the α -polyglutamated methotrexate compositions provided herein have lower toxic side effects compared to methotrexate. In some embodiments, the α -polyglutamated methotrexate compositions provided herein cause fewer or less severe side effects in vivo assays than methotrexate. In some embodiments, the in vivo assay is an in vivo murine model. In some embodiments, the α -polyglutamated methotrexate compositions provided herein cause fewer or less severe hematologic or hepatotoxic side effects than methotrexate. In some embodiments, hematological side effects are assessed by measuring mean neutrophil, mean leukocyte, or mean platelet counts. In some embodiments, hepatotoxic side effects are assessed by measuring serum aspartate transaminase (AST), serum alanine transaminase (ALT), and/or serum albumin levels. In some embodiments, the in vivo assay comprises administering 40mg/kg or 80mg/kg of the alpha polyglutamated methotrexate composition once per week for 4 weeks. In some embodiments, the alpha polyglutamated methotrexate is hexaglutamated methotrexate.
In some embodiments, treatment with the α polyglutamated methotrexate compositions provided herein does not induce significant hematologic or hepatotoxic side effects in an in vivo murine model. In some embodiments, hematological side effects are assessed by measuring mean neutrophil, mean leukocyte, or mean platelet counts. In some embodiments, hepatotoxic side effects are assessed by measuring serum aspartate transaminase (AST), serum alanine transaminase (ALT), and/or serum albumin levels. In some embodiments, the alpha polyglutamated methotrexate compositions provided herein do not significantly reduce mean neutrophil, mean leukocyte, or mean platelet counts. In some embodiments, the α -polyglutamated methotrexate compositions provided herein do not significantly increase serum aspartate transaminase (AST) and serum alanine transaminase (ALT) levels. In some embodiments, the α polyglutamated methotrexate compositions provided herein do not significantly reduce serum albumin levels. In some embodiments, the in vivo assay comprises administering 40mg/kg or 80mg/kg of the alpha polyglutamated methotrexate composition once per week for 4 weeks. In some embodiments, the alpha polyglutamated methotrexate is hexaglutamated methotrexate.
In some embodiments, the alpha-polyglutamated methotrexate composition is free of fluorine atoms. In some embodiments, the alpha polyglutamated methotrexate composition is free of 4-fluoro glutamyl.
Compositions of alpha polyglutamated methotrexate (alpha PMTX) and uses thereof are further described in each of U.S. application No. 62/374,458 and international application nos. PCT/US2017/046666 and PCT/US2017/046667, the contents of each of which are incorporated herein by reference in their entirety.
A. Polyglutamated methotrexate analogs and derivatives
The disclosure also encompasses alpha polyglutamated methotrexate derivatives and analogs. It is contemplated that the compositions and methods disclosed herein are applicable to any and every known derivative or analog of polyglutamated methotrexate. In some embodiments, polyglutamated methotrexate analog or derivative compositions made and used in accordance with the disclosed compositions and methods are depicted in figures 1I-1J. In some embodiments, the analog corresponds to a modified form of methotrexate in which the glutamyl groups of methotrexate are not linked to the remainder of the methotrexate molecule by gamma peptide bonds. In some embodiments, the analog is a variant form of methotrexate, wherein the glutamyl of methotrexate is in the D form. In some embodiments, the polyglutamated form of methotrexate or the polyglutamated methotrexate analog or derivative is not fluorinated.
In some embodiments, the polyglutamated methotrexate analogs or derivatives encompassed by the present disclosure are methotrexate derivatives bearing an indoline ring and a modified ornithine or glutamic acid. In some embodiments, the polyglutamated methotrexate analogs or derivatives encompassed by the present disclosure are members selected from the group consisting of: methotrexate derivatives carrying an indoline moiety, lipophilic amide methotrexate derivatives, methotrexate derivatives carrying an alkyl-substituted phenyl ring C, polymeric cisplatin methotrexate derivatives, N- (L- α -aminoacyl) methotrexate derivatives, halogenated methotrexate derivatives, 7-methyl methotrexate derivatives, N- (ac-aminoacyl) methotrexate derivatives, biotin methotrexate derivatives, dichloromethotrexate and lipophilic methotrexate derivatives, methotrexate derivatives carrying a benzoxazine or benzothiazine moiety, and N-acyl-N α - (4-amino-4-deoxypteroyl) -L-ornithine derivatives,
in some embodiments, the polyglutamated methotrexate analogs or derivatives encompassed by the present disclosure are members selected from the group consisting of: deoxyuridylate methotrexate derivatives, 10-deazaaminopterin analogs, 5-deazaaminopterin or 10-deazaaminopterin (10-EDAM) analogs, 5, 10-deazaaminopterin methotrexate analogs, 8-alkyl-7, 8-dihydro analogs, methotrexate analogs containing L-threo- (2S,4S) -4-fluoroglutamic acid or DL-3, 3-difluoroglutamic acid, methotrexate tetrahydroquinazoline analogs, D-glutamic methotrexate analogs, D-erythro forms, threo-4-fluoroglutamic methotrexate analogs, β γ -endomethylene methotrexate analogs, γ -tetrazole methotrexate analogs, ortho isomers of aminopterin, hydroxymethyl methotrexate, D-, Gamma-fluoro methotrexate, gem-diphosphonic acid methotrexate analogs, alpha or gamma-substituted methotrexate analogs, 5-methyl-5-deazamethotrexate analogs, 8 deazamethotrexate analogs, acivicin methotrexate analogs, phosphonoglutamic acid analogs, poly (L-lysine) methotrexate conjugates, dilysine or trilysine methotrexate derivatives, methotrexate-gamma-dimyristoyl phosphatidylethanolamine, iodoacetyl lysine methotrexate analogs, methotrexate analogs containing 2, omega-diaminoalkanoic acids, methyl-5-deazaanalogs, quinazoline methotrexate analogs, pyrazine methotrexate analogs, cysteine or homocysteine methotrexate analogs, gamma-tert-butyl methotrexate ester, Methotrexate analogs, folate methotrexate analogs, 7-hydroxymethotrexate, poly-gamma-glutamyl methotrexate analogs, 3',5' -dichloromethotrexate, diazoketone and chloromethyl ketone methotrexate analogs, 10-propargylaminopterin or alkyl methotrexate analogs, methotrexate lectin derivatives, 3',5' -dichloromethotrexate, deazaaminopterin analogs, cysteine and homocysteine methotrexate analogs, and MX 068.
In a further embodiment, the alpha polyglutamated methotrexate derivative or analog has a variant polyglutamate chain. In some embodiments, the polyglutamate chains contain one or more natural or synthetic residues other than glutamate. In some embodiments, the polyglutamate chains contain one or more glutamyl groups that do not contain an amide linkage. In other embodiments, one or more glutamyl groups of the polyglutamate chain are derivatized.
Synthesis of MTX-PG
The methotrexate polyglutamate compositions provided herein can be obtained by the following synthetic procedures known in the art. Procedures for the synthesis of methotrexate, including various pharmaceutically acceptable salts or acids (e.g., disodium methotrexate) as well as crystalline and amorphous forms, and intermediates used in the synthesis of methotrexate include, but are not limited to, those described in U.S. patent nos. 2,512,572, 3,892,801, 3,989,703, 4,057,548, 4,067,867, 4,079,056, 4,080,325, 4,106,488, 4,136,101, 4,224,446, 4,306,064, 4,374,987, 4,421,913, 4,558,690, 4,662,359 and 4,767,859, and Calvert, semin.
Glutamyl residues can be added to the glutamyl residues of methotrexate using synthetic procedures known in the art. In some embodiments, the glutamyl residues are added sequentially to the glutamyl residues of methotrexate. In further embodiments, polyglutamate is added to the glutamyl residues of methotrexate using a "click chemistry" method or other bioconjugate chemistry known to those skilled in the art. Alternatively, a peptide of a desired length of glutamyl residues can be produced and added to a precursor of methotrexate that does not have glutamyl residues. Peptides may be produced using synthetic methods known in the art. In some embodiments, the initial glutamyl residue is bonded to the Wang resin, and additional glutamyl residues are added sequentially via solid phase peptide synthesis using F-moc chemistry. After addition of the final glutamyl residue, the methotrexate precursor is coupled to the peptide and the molecule is cleaved from the resin.
C. methotrexate-PG complex
The present inventors have surprisingly found that polyglutamated antifolates such as polyglutamated methotrexate (α PMTX) are capable of forming complexes with other compositions comprising therapeutic agents, including cytotoxic compounds, such as platinum-based compounds. Thus, in some embodiments, the present disclosure provides a complex of an alpha PMTX (e.g., an alpha PMTX disclosed herein) and a therapeutic agent or a salt or acid thereof.
In some embodiments, the alpha PMTX/complex comprises alpha PMTX and a therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic compound, such as a chemotherapeutic agent. In other embodiments, the alpha PMTX/complex contains a platinum-based drug, such as a platinum-based chemotherapeutic agent (e.g., cisplatin, carboplatin, and oxaliplatin). In other embodiments, the alpha PMTX/complex contains a taxane-based chemotherapeutic agent (e.g., paclitaxel and docetaxel). In other embodiments, the alpha PMTX/complex contains a cyclodextrin. In other embodiments, the alpha PMTX/complex is encapsulated in a liposome.
In some embodiments, the present disclosure provides a composition comprising a complex of alpha PMTX and a therapeutic agent or a salt or acid thereof. In other embodiments, the alpha PMTX/therapeutic agent complex comprises one or more alpha PMTX containing 2-150, 2-100, 2-75, 2-50, 2-24, 2-30, 2-20, 2-19, 2-15, 2-10, or 2-5 glutamyl groups. In some embodiments, the alpha PMTX/therapeutic agent complex comprises one or more alpha PMTX comprising 3-10, 3-9, 3-8, or 3-7 glutamyl groups, or any range therebetween. In other embodiments, the alpha PMTX/therapeutic agent complex comprises one or more alpha PMTX comprising 4-10, 4-9, 4-8, 4-7, 4-6, or 4-5 glutamyl groups, or any range therebetween. In a particular embodiment, the complex comprises one or more alpha PMTX containing 3-10 glutamyl groups. In other embodiments, the alpha PMTX/therapeutic agent complex comprises one or more alpha PMTX containing 3-7 glutamyl groups. In another embodiment, the alpha PMTX/therapeutic agent complex comprises one or more alpha PMTX containing 5 glutamyl groups. In another embodiment, the alpha PMTX/therapeutic agent complex comprises one or more alpha PMTX containing 6 glutamyl groups. In some embodiments, the therapeutic agent is a cytotoxic compound or a salt or acid thereof. In another embodiment, the therapeutic agent is a chemotherapeutic agent or a salt or acid thereof. In another embodiment, the therapeutic agent is a platinum-based drug. In another embodiment, the therapeutic agent is a taxane-based drug. In further embodiments, the molar ratio of alpha PMTX/therapeutic agent in the complex is in the range of 1-10: 1. In some embodiments, the molar ratio of alpha PMTX/therapeutic agent in the complex is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1 (21-50), or 1: > 50. In some embodiments, the molar ratio of alpha PMTX// therapeutic agent in the complex is: 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1 or >50: 1. In some embodiments, the alpha PMTX/therapeutic agent complex is encapsulated in a liposome (e.g., as described herein or otherwise known in the art).
In another embodiment, the alpha PMTX complex comprises alpha PMTX and cyclodextrin. In some embodiments, the molar ratio of alpha PMTX (e.g., alpha PMTX salt)/cyclodextrin in the complex is in the range of 1-20:1, or any range therebetween. In some embodiments, the molar ratio of alpha PMTX/cyclodextrin in the complex is in the range of 1-10:1, or any range therebetween. In other embodiments, the molar ratio of alpha PMTX/cyclodextrin in the complex is in the range of 2-8:1, or any range therebetween. In some embodiments, the molar ratio of alpha PMTX/cyclodextrin in the complex is: 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1 or >50: 1. In other embodiments, the molar ratio of alpha PMTX/cyclodextrin in the complex is in the range of 1:1 to 20, 1:1 to 10, or 1:2 to 8, or any range therebetween. In some embodiments, the molar ratio of alpha PMTX/cyclodextrin in the complex is: 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1 (21-50), or 1: > 50. In some embodiments, the alpha PMTX/cyclodextrin complex is encapsulated in a liposome (e.g., as described herein or otherwise known in the art).
In some embodiments, the present disclosure provides a composition comprising an alpha PMTX/platinum-based chemotherapeutic agent complex. In some embodiments, the platinum-based chemotherapeutic agent is selected from the group consisting of: cisplatin, carboplatin, and oxaliplatin, or salts or acids thereof. In other embodiments, the alpha PMTX/platinum-based chemotherapeutic complex comprises an analog of cisplatin, carboplatin, oxaliplatin, or a salt or acid thereof. In some embodiments, the mole ratio of alpha PMTX/platinum-based agent in the composite is in the range of 1-20:1, or any range therebetween. In some embodiments, the mole ratio of alpha PMTX/platinum-based agent in the composite is in the range of 1-10:1, or any range therebetween. In other embodiments, the mole ratio of alpha PMTX/platinum-based agent in the composite is in the range of 2-8:1, or any range therebetween. In some embodiments, the molar ratio of alpha PMTX/platinum-based agent in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50: 1, or >50: 1. In other embodiments, the molar ratio of alpha PMTX/platinum-based chemotherapeutic agent in the complex is in the range of 1:1 to 20, 1:1 to 10, or 1:2 to 8, or any range therebetween. In some embodiments, the mole ratio of alpha PMTX/platinum-based agent in the composite is: 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1 (21-50), or 1: > 50. In further embodiments, the alpha PMTX// platinum-based agent complex is encapsulated in a liposome (e.g., as described herein or otherwise known in the art).
In additional embodiments, the alpha PMTX/platinum-based chemotherapeutic complex comprises an analog of cisplatin, carboplatin, oxaliplatin, or a salt or acid thereof. In some embodiments, the mole ratio of alpha PMTX/platinum-based analog in the composite is in the range of 1-20:1, or any range therebetween. In some embodiments, the mole ratio of alpha PMTX/platinum-based analog in the composite is in the range of 1-10:1, or any range therebetween. In other embodiments, the mole ratio of alpha PMTX/platinum-based agent in the composite is in the range of 2-8:1, or any range therebetween. In some embodiments, the molar ratio of alpha PMTX/platinum-based analog in the complex is 11:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50: 1, or >50: 1. In some embodiments, the mole ratio of alpha PMTX/platinum-based agent in the composite is: 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1 (21-50), or 1: > 50. In further embodiments, the alpha PMTX// platinum-based analog complex is encapsulated in a liposome (e.g., as described herein or otherwise known in the art).
In other embodiments, the present disclosure provides a complex comprising alpha PMTX and cisplatin, or a salt or acid thereof. In some embodiments, the molar ratio of alpha PMTX/cisplatin (or cisplatin salt or acid) in the complex is in the range of 1-20:1, or any range therebetween. In some embodiments, the molar ratio of alpha PMTX/cisplatin (or cisplatin salt or acid) in the complex is in the range of 1-10:1, or any range therebetween. In other embodiments, the molar ratio of alpha PMTX/cisplatin (or cisplatin salt or acid) in the complex is in the range of 2-8:1, or any range therebetween. In some embodiments, the molar ratio of alpha PMTX/cisplatin (or cisplatin salt or acid) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50: 1) or >50: 1. In some embodiments, the molar ratio of alpha PMTX/cisplatin (or cisplatin salt or acid) in the complex is: 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1 (21-50), or 1: > 50. In further embodiments, the alpha PMTX// cisplatin (or cisplatin salt or acid) complex is encapsulated in a liposome (e.g., as described herein or otherwise known in the art).
In another embodiment, the present disclosure provides a complex comprising alpha PMTX and carboplatin or a salt or acid thereof. In some embodiments, the mole ratio of alpha PMTX/carboplatin (or carboplatin salt or acid) in the composite is in the range of 1-20:1, or any range therebetween. In other embodiments, the mole ratio of alpha PMTX/carboplatin (or carboplatin salt or acid) in the composite is in the range of 1-10:1, or any range therebetween. In other embodiments, the mole ratio of alpha PMTX/carboplatin (or carboplatin salt or acid) in the composite is in the range of 2-8:1, or any range therebetween. In some embodiments, the molar ratio of alpha PMTX/carboplatin (or carboplatin salt or acid) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1, or >50: 1. In some embodiments, the molar ratio of alpha PMTX/cyclodextrin in the complex is: 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1 (21-50), or 1: > 50. In further embodiments, the alpha PMTX/carboplatin (or carboplatin salt or acid) complex is encapsulated in a liposome (e.g., as described herein or otherwise known in the art).
In another embodiment, the present disclosure provides a complex containing alpha PMTX and oxaliplatin or a salt or acid thereof. In some embodiments, the molar ratio of alpha PMTX/oxaliplatin (or oxaliplatin salt or acid) in the complex is in the range of 1-20:1, or any range therebetween. In other embodiments, the molar ratio of alpha PMTX/oxaliplatin (or oxaliplatin salt or acid) in the complex is in the range of 1-10:1, or any range therebetween. In other embodiments, the molar ratio of alpha PMTX/oxaliplatin (or oxaliplatin salt or acid) in the complex is in the range of 2-8:1, or any range therebetween. In some embodiments, the molar ratio of alpha PMTX/oxaliplatin (or oxaliplatin salt or acid) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50: 1, or >50: 1. In some embodiments, the molar ratio of alpha PMTX/oxaliplatin (or oxaliplatin salt or acid) in the complex is: 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1 (21-50), or 1: > 50. In further embodiments, the alpha PMTX/oxaliplatin (or oxaliplatin salt or acid) complex is encapsulated in a liposome (e.g., as described herein or otherwise known in the art).
In additional embodiments, the present disclosure provides a complex comprising alpha PMTX and a platinum-based chemotherapeutic agent ("platinum") selected from the group consisting of: nedaplatin, heptaplatin, lobaplatin, satraplatin, carboplatin (paratoplatin), cisplatin (platinol), cycloplatin, dexormaplatin, spiroplatin, picoplatin, triplatin, tetraplatin, iproplatin, ormaplatin, zeniplatin, platinum-triamine, traplatin, enloplatin, JM216, NK121, CI973, DWA 2114R, NDDP, and dedaplatin, or salts or acids thereof. In other embodiments, the alpha PMTX/platinum-based chemotherapeutic complex comprises nedaplatin, heptaplatin, lobaplatin, satraplatin, carboplatin, cisplatin, cycloplatin, dexormaplatin, spiroplatin, picoplatin, triplatin, tetraplatin, iproplatin, ormaplatin, zeniplatin, platinum-triamine, traplatin, enloplatin, JM216, NK121, CI973, DWA 2114R, NDDP, or an analog of dedaplatin, or a salt or acid thereof. In some embodiments, the molar ratio of alpha PMTX/platinum (or platinum salt or acid) in the composite is in the range of 1-20:1, or any range therebetween. In other embodiments, the molar ratio of alpha PMTX/platinum (or platinum salt or acid) in the composite is in the range of 1-10:1, or any range therebetween. In other embodiments, the molar ratio of alpha PMTX/platinum (or platinum salt or acid) in the composite is in the range of 2-8:1, or any range therebetween. In some embodiments, the molar ratio of alpha PMTX/platinum (or platinum salt or acid) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50: 1, or >50: 1. In some embodiments, the molar ratio of alpha PMTX/platinum (or platinum salt or acid) in the composite is: 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1 (21-50), or 1: > 50. In further embodiments, the alpha PMTX/platinum (or salt or acid or analog thereof) complex is encapsulated in a liposome (e.g., as described herein or otherwise known in the art).
In some embodiments, the present disclosure provides a composition comprising an alpha PMTX/taxane-based chemotherapeutic (taxane) complex. In some embodiments, the taxane-based chemotherapeutic agent is selected from the group consisting of: paclitaxel (PTX), Docetaxel (DTX), Larotaxel (LTX) and Cabazitaxel (CTX), or salts or acids thereof. In some embodiments, the molar ratio of alpha PMTX/taxane-based agent in the complex is in the range of 1-20:1, or any range therebetween. In other embodiments, the molar ratio of alpha PMTX/taxane (or taxane salt or acid) in the complex is in the range of 1-10:1, or any range therebetween. In other embodiments, the molar ratio of alpha PMTX/taxane (or taxane salt or acid) in the complex is in the range of 2-8:1, or any range therebetween. In some embodiments, the molar ratio of alpha PMTX/taxane (or taxane salt or acid) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50: 1 or >50: 1. In some embodiments, the molar ratio of alpha PMTX/taxane (or taxane salt or acid) in the complex is: 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1 (21-50), or 1: > 50. In additional embodiments, the alpha PMTX/taxane-based agent complex is encapsulated in a liposome (e.g., as described herein or otherwise known in the art).
In additional embodiments, the present disclosure provides a complex comprising alpha PMTX and Paclitaxel (PTX) or a salt or acid thereof. In other embodiments, the alpha PMTX/taxane-based chemotherapeutic complex comprises an analog of Paclitaxel (PTX) or a salt or acid thereof. In some embodiments, the molar ratio of alpha PMTX/paclitaxel (or paclitaxel salt or acid) in the complex is in the range of 1-20:1, or any range therebetween. In other embodiments, the molar ratio of alpha PMTX/paclitaxel (or paclitaxel salt or acid) in the complex is in the range of 1-10:1, or any range therebetween. In other embodiments, the molar ratio of alpha PMTX/paclitaxel (or paclitaxel salt or acid) in the complex is in the range of 2-8:1, or any range therebetween. In some embodiments, the molar ratio of alpha PMTX/paclitaxel (or paclitaxel salt or acid) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50: 1) or >50: 1. In some embodiments, the molar ratio of α PMTX/paclitaxel (or paclitaxel salt or acid) in the complex is: 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1 (21-50), or 1: > 50. In further embodiments, the alpha PMTX/paclitaxel (or paclitaxel salt or acid) complex is encapsulated in a liposome (e.g., as described herein or otherwise known in the art).
In additional embodiments, the present disclosure provides a complex comprising alpha PMTX and Docetaxel (DTX) or salts or acids thereof. In other embodiments, the alpha PMTX/taxane-based chemotherapeutic complex comprises an analog of Docetaxel (DTX) or a salt or acid thereof. In some embodiments, the molar ratio of alpha PMTX/docetaxel (or docetaxel salt or acid) in the complex is in the range of 1-20:1, or any range therebetween. In some embodiments, the molar ratio of alpha PMTX/docetaxel (or docetaxel salt or acid) in the complex is in the range of 1-10:1, or any range therebetween. In other embodiments, the molar ratio of alpha PMTX/docetaxel (or docetaxel salt or acid) in the complex is in the range of 2-8:1, or any range therebetween. In some embodiments, the molar ratio of alpha PMTX/docetaxel (or docetaxel salt or acid) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50: 1 or >50: 1. In some embodiments, the molar ratio of alpha PMTX/docetaxel (or docetaxel salt or acid) in the complex is: 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1 (21-50), or 1: > 50. In further embodiments, the alpha PMTX/docetaxel (or docetaxel salt or acid) complex is encapsulated in a liposome (e.g., as described herein or otherwise known in the art).
In additional embodiments, the present disclosure provides a complex comprising alpha PMTX and raloxitol (LTX) or a salt or acid thereof. In other embodiments, the alpha PMTX/taxane-based chemotherapeutic complex comprises an analog of ralfataxel (LTX) or a salt or acid thereof. In some embodiments, the molar ratio of alpha PMTX/raloxitol (or raloxitol salt or acid) in the composite is in the range of 1-20:1, or any range therebetween. In other embodiments, the molar ratio of alpha PMTX/raloxitol (or raloxitol salt or acid) in the composite is in the range of 1-10:1, or any range therebetween. In other embodiments, the molar ratio of alpha PMTX/raloxitol (or raloxitol salt or acid) in the composite is in the range of 2-8:1, or any range therebetween. In some embodiments, the molar ratio of alpha PMTX/raloxitol (or raloxitol salt or acid) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50: 1, or >50: 1. In some embodiments, the molar ratio of alpha PMTX/raloxitol (or raloxitol salt or acid) in the composite is: 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1 (21-50), or 1: > 50. In additional embodiments, the alpha PMTX/raloxitol (or raloxitol salt or acid) complex is encapsulated in a liposome (e.g., as described herein or otherwise known in the art).
In additional embodiments, the present disclosure provides a complex comprising alpha PMTX and Cabazitaxel (CTX) or salts or acids thereof. In other embodiments, the alpha PMTX/taxane-based chemotherapeutic complex comprises an analog of Cabazitaxel (CTX) or a salt or acid thereof. In some embodiments, the molar ratio of alpha PMTX/cabazitaxel (or cabazitaxel salt or acid) in the composite is in the range of 1-20:1, or any range therebetween. In other embodiments, the molar ratio of alpha PMTX/cabazitaxel (or cabazitaxel salt or acid) in the composite is in the range of 1-10:1, or any range therebetween. In other embodiments, the molar ratio of alpha PMTX/cabazitaxel (or cabazitaxel salt or acid) in the composite is in the range of 2-8:1, or any range therebetween. In some embodiments, the molar ratio of α PMTX/cabazitaxel (or cabazitaxel salt or acid) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50: 1, or >50: 1. In some embodiments, the molar ratio of alpha PMTX/cabazitaxel (or cabazitaxel salt or acid) in the complex is: 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1 (21-50), or 1: > 50. In additional embodiments, the alpha PMTX/cabazitaxel (or cabazitaxel salt or acid) complex is encapsulated in a liposome (e.g., as described herein or otherwise known in the art).
In a further embodiment, the present disclosure provides a complex comprising alpha PMTX and another antimetabolite, or a salt or acid thereof. An antimetabolite is a chemical substance that has a structure similar to a metabolite required for normal biochemical reactions, but differs from the metabolite sufficiently to interfere with one or more normal functions of a cell (such as cell division). In some embodiments, the present disclosure provides a complex comprising alpha PMTX and Methotrexate (MTX), or a salt or acid thereof. In some embodiments, the present disclosure provides a complex comprising alpha PMTX and an antimetabolite selected from the group consisting of: gemcitabine, fluorouracil, capecitabine, antifolates (e.g., methotrexate), tegafur, cytarabine, thioguanine, 5-azacytidine, 6-mercaptopurine, azathioprine, 6-thioguanine, pentostatin, fludarabine phosphate, and cladribine, as well as pharmaceutically acceptable salts or acids, or derivatives of any of these. In some embodiments, the molar ratio of alpha PMTX/antimetabolite (or antimetabolite salt or acid) in the complex is in the range of 1-20:1, or any range therebetween. In other embodiments, the molar ratio of alpha PMTX/antimetabolite (or antimetabolite salt or acid) in the complex is in the range of 1-10:1, or any range therebetween. In other embodiments, the molar ratio of alpha PMTX/antimetabolite (or antimetabolite salt or acid) in the complex is in the range of 2-8:1, or any range therebetween. In some embodiments, the molar ratio of alpha PMTX/antimetabolite (or antimetabolite salt or acid) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50: 1) or >50: 1. In some embodiments, the molar ratio of alpha PMTX/antimetabolite (or antimetabolite salt or acid) in the complex is 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1 (21-50), or 1: > 50. In additional embodiments, the alpha PMTX/antimetabolite (or antimetabolite salt or acid) complex is encapsulated in a liposome (e.g., as described herein or otherwise known in the art).
In additional embodiments, the present disclosure provides a complex of alpha PMTX (e.g., an alpha PMTX disclosed herein) and a cyclodextrin. Cyclodextrins (CDs) are groups of cyclic oligosaccharides that have been shown to improve the physicochemical properties of many drugs by forming complexes. CD is a cyclic oligosaccharide consisting of several D-glucose units linked by alpha- (1,4) bonds. This annular configuration provides a hydrophobic interior cavity and renders the CD frustoconical. Many hydroxyl groups are located at the edge of the ring, which makes CD lipophilic and water soluble. As a result, CD is able to form complexes with a wide variety of hydrophobic agents and thus alter the physico-chemical properties of these complexing agents.
Unless otherwise indicated herein, the term "cyclodextrin" or "CD" generally refers to a parent or derived cyclic oligosaccharide containing a variable number of (α -1,4) -linked D-glucopyranoside units, which is capable of forming a complex with methotrexate-PG. Each cyclodextrin glucopyranoside subunit has secondary hydroxyl groups at the 2 and 3 positions, and a primary hydroxyl group at the 6 position. The term "parent", "underivatized" or "inert" cyclodextrin is meant to contain a cyclodextrin having the basic formula C6H12O6And a glucose structure without any additional chemically substituted cyclodextrin (e.g., an α -cyclodextrin consisting of 6D-glucopyranoside units, a β -cyclodextrin consisting of 7D-glucopyranoside units, and a γ -cyclodextrin consisting of 8D-glucopyranoside units). The physical and chemical properties of the parent cyclodextrins can be modified by derivatizing the hydroxyl groups with other functional groups. Any substance that is located within the internal phase of a cyclodextrin is said to "complex" with the cyclodextrin, or form a complex (inclusion complex) with the cyclodextrin.
As used herein, there is no particular limitation on the cyclodextrin component of the α PMTX/cyclodextrin complex, as long as the cyclodextrin can form a complex with the α PMTX. In particular embodiments, the cyclodextrin has been derivatized to carry ionizable (e.g., weakly basic and/or weakly acidic) functional groups to facilitate complex formation with alpha PMTX and/or liposome encapsulation.
It is known that modifying the hydroxyl groups of cyclodextrins with ionizable chemical groups (e.g., those that face away from the cyclodextrin internal phase) can facilitate loading of cyclodextrins and therapeutic agents complexed with cyclodextrins. In some embodiments, the cyclodextrin of the alpha PMTX/cyclodextrin complex has at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 hydroxyl groups substituted with an ionizable chemical group. Term "By charged cyclodextrin "is meant a cyclodextrin in which one or more of its hydroxyl groups is replaced with a charged moiety. Such a moiety may itself be a charged group, or it may comprise an organic moiety substituted with one or more charged moieties (e.g., C)1-C6Alkyl or C1-C6Alkyl ether moieties).
In some embodiments, the "ionizable" or "charged" moiety of the CD derivative is weakly ionizable. Weakly ionizable moieties are those moieties that are weakly basic or weakly acidic. According to CH3-W, the weakly basic functional group (W) has a pKa between about 6.0-9.0, 6.5-8.5, 7.0-8.0, 7.5-8.0, and any range therebetween (inclusive). Similarly, the weak acidic functional group (X) has a logarithmic dissociation constant (pKa) according to CH3-X of between about 3.0-7.0, 4.0-6.5, 4.5-6.5, 5.0-6.0, 5.0-5.5, and any range therebetween (inclusive). Representative anionic moieties include, but are not limited to, carboxylate, carboxymethyl, succinyl, sulfonyl, phosphate, sulfoalkyl ether, sulfatocarbonate, thiocarbonate, dithiocarbonate, phosphate, phosphonate, sulfonate, nitrate, and borate groups. Representative cationic moieties include, but are not limited to, amino, guanidine, and quaternary ammonium groups.
In another embodiment, the derivatized cyclodextrin is a "polyanion" or "polycation". Polyanions are derivatized cyclodextrins having more than one negatively charged group resulting in a net negative ionic charge of more than two units. Polycations are derivatized cyclodextrins that have more than one positively charged group, resulting in a net positive ionic charge of more than two units.
In another embodiment, the derivatized cyclodextrin is a "chargeable amphiphile". By "chargeable" is meant that the pK of the amphiphile is in the range of pH 4 to pH 8 or 8.5. Thus, the chargeable amphiphile may be a weak acid or a weak base. "amphoteric" herein refers to a derivatized cyclodextrin having ionizable groups of both anionic and cationic character, wherein: (a) at least one and optionally both of the cationic and anionic amphiphiles are chargeable, having at least one charged group with a pK between 4 and 8 to 8.5; (b) the cationic charge predominates at pH 4, and (c) the anionic charge predominates at pH 8 to 8.5.
In some embodiments, the derivatized cyclodextrin, whether polyionic, amphiphilic, or otherwise, "ionizable" or "charged" as a whole is weakly ionizable (i.e., has a pKai between about 4.0-8.5, 4.5-8.0, 5.0-7.5, 5.5-7.0, 6.0-6.5, and any range therebetween, inclusive)).
Any, some, or all of the hydroxyl groups in any, some, or all of the α -D-glucopyranoside units of the cyclodextrin can be modified to an ionizable chemical group as described herein. Because of the different chemical reactivity of each cyclodextrin hydroxyl group, reaction with the modifying moiety can produce an amorphous mixture of positional and optical isomers. Alternatively, certain chemical methods can react a pre-modified α -D-glucopyranoside unit to form a homogeneous product.
The aggregation substitution that occurs with cyclodextrin derivatives in a mixture is described in terms of the degree of substitution. For example, a 6-ethylenediamino- β -cyclodextrin having a degree of substitution of seven would consist of an isomeric distribution of 6-ethylenediamino- β -cyclodextrin in which the average number of ethylenediamino groups per 6-ethylenediamino- β -cyclodextrin molecule is seven. The degree of substitution of the mixture of cyclodextrin derivatives can be routinely determined using mass spectrometry or nuclear magnetic resonance spectrometry.
In one embodiment, at least one hydroxyl moiety facing away from the cyclodextrin interior is substituted with an ionizable chemical group. For example, the C2, C3, C6, C2 and C3, C2 and C6, C3 and C6, and all three C2-C3-C6 hydroxyl groups of at least one α -D-glucopyranoside unit are substituted with an ionizable chemical group. Any such combination of hydroxyl groups can be similarly combined with at least two, three, four, five, six, seven, eight, nine, ten, eleven, up to all of the α -D-glucopyranoside units in the modified cyclodextrin, as well as with any degree of substitution described herein. One such derivative is sulfoalkyl ether cyclodextrin (SAE-CD). Sulfobutyl ether derivatives of beta cyclodextrin (SBE-beta-CD) have demonstrated significantly improved water solubility compared to the parent cyclodextrin.
Additional cyclodextrin derivatives that can be complexed with the therapeutic agent in the disclosed liposome compositions include sugammadex or Org-25969, wherein the 6-hydroxy group on the gamma-CD has been replaced with a carboxythioacetate ether linkage and a hydroxybutenyl-beta-CD. Alternatives to cyclodextrins include: 2, 6-di-O-methyl- β -CD (DIMEB), 2-hydroxypropyl-3-cyclodextrin (HP- β -CD), randomly methylated- β -cyclodextrin (RAMEB), sulfobutyl ether β -cyclodextrin (SBE- β -CD), and sulfobutyl ether- γ -cyclodextrin (SBE γ CD), sulfobutylated β -cyclodextrin sodium salt, (2-hydroxypropyl) - α -cyclodextrin, (2-hydroxypropyl) - β -cyclodextrin, (2-hydroxypropyl) - γ -cyclodextrin, 2, 6-di-O-methyl) - β -cyclodextrin (DIMEB-50 hepta), 2,3, 6-tri-O-methyl) - β -cyclodextrin (TRIMEB hepta), Methyl-beta-cyclodextrin, octa (6-deoxy-6-iodo) -gamma-cyclodextrin, and octa (6-deoxy-6-bromo) -gamma-cyclodextrin.
In some embodiments, the one or more cyclodextrins have a high solubility in water, such that a greater amount of the cyclodextrins is entrapped in the liposome internal phase. In some embodiments, the water solubility of the cyclodextrin is at least 10mg/mL, 20mg/mL, 30mg/mL, 40mg/mL, 50mg/mL, 60mg/mL, 70mg/mL, 80mg/mL, 90mg/mL, 100mg/mL or higher. In some embodiments, the water solubility of the one or more cyclodextrins is in the range of 10-150mg/mL, 20-100mg/mL, 20-75mg/mL, and any range in between (including the endpoints).
In some embodiments, large association constants between cyclodextrin and α PMTX and/or other therapeutic agents complexed with cyclodextrin are preferred and can be obtained by selecting the number of glucose units in cyclodextrin based on the size of the therapeutic agent (see, e.g., Albers et al, crit. rev. therapy. drug Carrier system.12: 311-337 (1995); Stella et al, toxicol. pathol.36:30-42 (2008.) when the association constant is dependent on pH, cyclodextrin can be selected such that the association constant becomes larger at pH in the liposome phase 200. 200-1,000, 300-750, and any range therebetween.
In some embodiments, the cyclodextrin of the alpha PMTX/cyclodextrin complex and/or the cyclodextrin/therapeutic agent complex is underivatized.
In some embodiments, the cyclodextrin of the alpha PMTX/cyclodextrin complex and/or the cyclodextrin/therapeutic agent complex is derivatized. In other embodiments, the cyclodextrin derivative of the complex has the structure of formula I:
Figure BDA0002715283060000461
Wherein: n is 4, 5 or 6;
wherein R is1、R2、R3、R4、R5、R6、R7、R8And R9Each independently is-H, straight or branched chain C1-C8Alkylene or optionally substituted straight or branched C1-C6Group, wherein R1、R2、R3、R4、R5、R6、R7、R8And R9At least one of which is a straight or branched chain C1-C8Alkylene (e.g. C)1-C8- (alkylene) -SO3 -A group);
in some embodiments, the cyclodextrin derivative of the alpha PMTX/cyclodextrin complex and/or the cyclodextrin/therapeutic agent complex has the structure of formula II:
Figure BDA0002715283060000462
wherein: n is 4, 5 or 6;
wherein R is1、R2、R3、R4、R5、R6、R7、R8And R9Each is independentThe root is-O-or-O- (C)2-C6Alkylene) -SO3-a group; wherein R is1And R2At least one of which is independently-O- (C)2-C6Alkylene) -SO3 -A group; and S1、S2、S3、S4、S5、S6、S7、S8And S9Each independently is a pharmaceutically acceptable cation. In other embodiments, the pharmaceutically acceptable cation is selected from: alkali metals, e.g. Li+、Na+Or K+(ii) a Alkaline earth metals, e.g. Ca+2Or Mg+2And ammonium ions and amine cations, such as the cations of (C1-C6) -alkylamines, piperidines, pyrazines, (C1-C6) -alkanolamines and (C4-C8) -cycloalkanolamines. In some embodiments, at least one of R1 and R2 is independently a-O- (C2-C6 alkylene) -SO 3-group that is-O- (CH)2)mA SO 3-group, wherein m is 2 to 6, preferably 2 to 4 (e.g., -O-CH 2S 03-or-O-CH 2S 03-); and S 1、S2、S3、S4、S5、S6、S7、S8And S9Each independently is H or a pharmaceutically acceptable cation including, for example, an alkali metal (e.g., Li)+、Na+、K+) Alkaline earth metal (e.g., Ca)+2、Mg+2) Ammonium ions and amine cations, e.g. (C1-C6) -alkylamines, piperidines, pyrazines, (C6)1-C6) -alkanol-amine and (C)4-C8) -cation of cycloalkanolamine:
in some embodiments, the cyclodextrin derivative of the alpha PMTX/cyclodextrin complex and/or the cyclodextrin/therapeutic agent complex is any one of U.S. patent nos. 6,133,248, 5,874,418, 6,046,177, 5,376,645, 5,134,127, 7,034,013, 6,869,939; and cyclodextrins disclosed in international application publication No. WO 02005/117911, the contents of each of which are incorporated herein by reference in their entirety for all purposes.
In some embodiments, the cyclodextrin derivative of the alpha PMTX/cyclodextrin complex and/or the cyclodextrin/therapeutic agent complex is a sulfoalkyl ether cyclodextrin. In some casesIn one embodiment, the cyclodextrin derivative of the complex is sulfobutyl ether-3-cyclodextrin, e.g.
Figure BDA0002715283060000463
(CyDex Pharma.Inc., Lenexa, Kansas. methods for preparing sulfobutyl ether-3-cyclodextrins and other sulfoalkyl ether cyclodextrins are known in the art.
In some embodiments, the cyclodextrin derivative in the alpha PMTX/cyclodextrin complex and/or the cyclodextrin/therapeutic agent complex is a compound of formula III:
Figure BDA0002715283060000471
Wherein R is equal to:
(a)(H)21-Xor (- (CH)2)4-SO3Na) x, and x ═ 1.0 to 10.0, 1.0 to 5.0, 6.0 to 7.0, or 8.0 to 10.0;
(b)(H)21-Xor (- (CH)2CH(OH)CH3) x, and x ═ 1.0 to 10.0, 1.0 to 5.0, 6.0 to 7.0, or 8.0 to 10.0;
(c)(H)21-Xor (sulfoalkyl ether) x, and x ═ 1.0 to 10.0, 1.0 to 5.0, 6.0 to 7.0, or 8.0 to 10.0; or
(d)(H)21-XOr (- (CH)2)4-SO3Na) x, and x ═ 1.0-10.0, 1.0-5.0, 6.0-7.0, or 8.0-10.0.
In additional embodiments, the alpha PMTX/cyclodextrin complex and/or cyclodextrin/therapeutic agent complex is encapsulated in a liposome (e.g., as described herein or otherwise known in the art).
α PMTX delivery vehicle
In alternative embodiments, the present disclosure provides an α PMTX delivery system and its use to deliver a payload of α PMTX to one or more cells in vitro or in vivo. In some embodiments, the α PMTX is complexed with or incorporated into a delivery vehicle. Such delivery vehicles are known in the art and include, but are not limited to, liposomes, lipid spheres, polymers, peptides, proteins, antibodies (e.g., ADCs, such as antibody-alpha PMTX conjugates), cellular components, cyclic oligosaccharides (e.g., cyclodextrins), nanoparticles (e.g., lipid nanoparticles, biodegradable nanoparticles, and core-shell nanoparticles), lipoprotein particles, and combinations thereof. In a particular embodiment, the delivery vehicle is a liposome. In other particular embodiments, the delivery vehicle is an antibody or antigen-binding antibody fragment.
A. Liposomes
In some embodiments, the present disclosure provides liposome compositions comprising liposomes encapsulating (i.e., filled with) alpha polyglutamated methotrexate (e.g., alpha PMTX disclosed herein). In some embodiments, the liposomes in the liposome composition comprise alpha PMTX containing 4, 5, 2-10, 4-6, or more than 5 glutamyl groups (including glutamyl groups in methotrexate). In some embodiments, the alpha polyglutamated methotrexate in the Lp-alpha PMTX comprises two or more glutamyl groups in the L form. In other embodiments, the α polyglutamated methotrexate in Lp- α PMTX comprises a glutamyl group in the D form. In other embodiments, the α polyglutamated methotrexate in the Lp- α PMTX comprises a glutamyl group in the D form and two or more glutamyl groups in the L form. In further embodiments, the α polyglutamated methotrexate in the Lp- α PMTX comprises two or more glutamyl groups with a γ carboxyl linkage. In some embodiments, the alpha polyglutamated methotrexate in the Lp-alpha PMTX comprises at least one glutamyl group having both an alpha carboxyl linkage and a gamma carboxyl linkage. In some embodiments, the liposome composition comprises liposomes comprising alpha pentaglutamated MTX. In other embodiments, the liposome comprises L-alpha pentaglutamated MTX, D-alpha pentaglutamated MTX, or L-and D-alpha pentaglutamated MTX. In some embodiments, the liposome composition comprises liposomes comprising alpha hexaglutaminated MTX (Lp-alpha PMTX). In other embodiments, the liposome comprises L-alpha hexaglutamated MTX, D-alpha hexaglutamated MTX, or L-and D-alpha hexaglutamated MTX. In some embodiments, the liposome composition comprises anionic or neutral liposomes. In some embodiments, the liposome composition comprises cationic liposomes. In some embodiments, the Lp-alpha PMTX composition is non-pegylated. In some embodiments, the Lp- α PMTX composition is non-targeted (NTLp- α PMTX). In other embodiments, the Lp-alpha PMTX composition is targeted (TLp-alpha PMTX). In some embodiments, the liposome composition comprises liposomes having a diameter in the range of 20nm to 500nm or any range therebetween. In some embodiments, the liposome composition comprises liposomes having a diameter in the range of 20nm to 400nm or any range therebetween. In some embodiments, the liposome composition comprises liposomes having a diameter in the range of 20nm to 300nm or any range therebetween. In some embodiments, the liposome composition comprises liposomes having a diameter in the range of 20nm to 200nm or any range therebetween. In other embodiments, the liposome composition comprises liposomes having a diameter in the range of 20nm to 150nm or any range therebetween. In other embodiments, the liposome composition comprises liposomes having a diameter in the range of 80nm to 120nm or any range therebetween. In further embodiments, 30% -70%, 30% -60%, or 30% -50% w/w or any range therebetween of α -polyglutamated methotrexate is encapsulated (embedded) in Lp- α PMTX. In some embodiments, at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75% of the alpha polyglutamated methotrexate is encapsulated in Lp-alpha PMTX during the preparation of the liposomes.
In some embodiments, provided liposomes further comprise an immunostimulant, a detectable label, or both disposed on the exterior thereof. The immunostimulatory agent or detectable label can be ionically or covalently bound to the exterior of the liposome, including, for example, a steric stabilizer component optionally bound to the liposome.
The term "immunostimulating agent", also known as "immunostimulant" and "immunostimulator" refers to a substance that stimulates immunity (including a pre-existing immune response) by inducing activation or increasing activity of any component of the immune system. These immune stimulators may include one or more of haptens, adjuvants, protein immune stimulators, nucleic acid immune stimulators, and chemical immune stimulators. Many adjuvants contain substances intended to stimulate an immune response, such as lipid a, proteins of bordetella pertussis or mycobacterium tuberculosis origin. Certain adjuvants are commercially available, for example, Freund's incomplete and complete adjuvants (Difco Laboratories, Detroit, Mich.); merck adjuvant 65(Merck and Company, inc., Rahway, n.j.); AS-2(SmithKline Beecham, Philadelphia, Pa.); aluminum salts such as aluminum hydroxide gel (alum) or aluminum phosphate; salts of calcium, iron or zinc; an insoluble suspension of acylated tyrosine; acylated sugars; a cationically or anionically derivatized polysaccharide; polyphosphazene; biodegradable microspheres; monophosphoryl lipid a and quil a; IFN-gamma, IFN-alpha, FLT 3-ligand; and immunostimulatory antibodies (e.g., anti-CTLA-4, anti-CD 28, anti-CD3. cytokines such as GM-CSF, interleukin-2, -7, -12, and-15, and other similar growth factors may also be used as adjuvants. in a preferred embodiment, the immunostimulatory agent may be at least one selected from the group consisting of fluorescein, DNP, beta glucan, beta-1, 3-glucan, beta-1, 6-glucan. in another preferred embodiment, the immunostimulatory agent is a Toll-like receptor (TLR) modulator. It surprisingly acts as an immunostimulant and detectable label based on our experiments.
In some embodiments, the liposome comprises a detectable marker. Detectable labels may, for example, include at least radioisotopes, fluorescent compounds, bioluminescent compounds, chemiluminescent compounds, metal chelators, enzymes, dyes, inks, magnetic compounds, biocatalysts, and pigments that are detectable by any suitable method known in the art, such as Magnetic Resonance Imaging (MRI), optical imaging, fluorescence/luminescence imaging, and/or nuclear imaging techniques.
In some embodiments, the immunostimulatory agent and/or detectable marker is attached to the exterior by co-incubating it with the liposome. For example, the immunostimulatory agent and/or detectable label may be associated with the liposome membrane by hydrophobic interactions or by ionic bonds such as avidin/biotin bonds or metal chelate bonds (e.g., Ni-NTA). Alternatively, the immunostimulant or detectable label may be covalently bonded to the exterior of the liposome, for example by covalent bonding to a liposome component or as a steric stabilizer for PEG.
In some embodiments, the liposome further comprises an agent that increases uptake of the liposome into a target cellular compartment that includes cytosol.
In some embodiments, the liposome comprises a mitochondrial targeting agent. In some embodiments, the liposome comprises Triphenylphosphonium (TPP). Methods and mechanisms for surface functionalization of liposomes with TPP are known in the art (e.g., TPP is linked to a lipid anchor via a peg spacer and TPP is modified with stearyl (stearyltriphenylphosphonium (STPP)). Comprising RQIKIWFQNRRMKWKKRKKRRQRRR (SEQ ID NO:1), RKKRRXR RRGC wherein X is any natural or non-natural amino acid (SEQ ID NO:2), CCGCCAAGAAGCG (SEQ ID NO:3), GCGTGCACACGCGCGTAGACTTCCCCCGCAAGTCACTCGTT AGCCCGCCAAGAAGCGACCCCTCCGGGGCGAGCTGAGCGGCGTGGCGCGGG GGCGTCAT (SEQ ID NO:4), ACGTGCATACGCACGTAGACATTCCCCGCTTCCCACTCCAAAGTCCGCCAAGAAGCGTATCCCGCTGAGCGGCGTGGCGCG GGGGCGTCATCCGTCAGCTC (SEQ ID NO:5) or ACTTCCCCCGCAAGTCAC TCGTTAGCCCGCCAAGAAGCGACCC CTCCGGGGCGAGCTG (SEQ ID NO:6)), or a mitochondrially penetrating fragment thereof.
In some embodiments, the liposomes in the provided liposome compositions comprise a mitochondrial penetrating agent selected from the group consisting of: guanidine-rich peptoids, tetraguanidines, triguanidines, bis-monoguanidines, guanidine-rich polyurethanes, beta-oligoarginine, proline-rich dendrimers, and phosphonium salts (e.g., methyltriphenylphosphonium and/or tetraphenylphosphonium).
In some embodiments, the liposomes in provided liposome compositions comprise sphingomyelin and/or stearyl-octa-arginine. In some embodiments, the liposome comprises sphingomyelin and/or stearyl-octa-arginine. In some embodiments, the liposome comprises DOPE, sphingomyelin, stearyl-octa-arginine sphingomyelin, and stearyl-octa-arginine. In some embodiments, the liposomes comprise DOPE, sphingomyelin, stearyl-octa-arginine sphingomyelin, and stearyl-octa-arginine in a 9:2:1 molar ratio. In some embodiments, the liposome comprises a MITO-porter system or a variant thereof.
In some embodiments, the liposomes in the provided liposome compositions comprise an agent, such as a cell penetrating agent, that facilitates delivery of the liposome across the cell membrane and provides the liposome with the ability to bypass the endocytic pathway and the harsh environment of the lysosome. Cell penetrating agents are known in the art and are routinely used and suitable for making and using the provided liposome compositions. In some embodiments, the cell penetrating/lysosomal bypassing agent is chloroquine. In some embodiments, the cell penetrating agent is a cell penetrating peptide. In some embodiments, the liposomes in the provided liposome compositions comprise a cell penetrating agent selected from the group consisting of: RKKRRQRRR (SEQ ID NO:7), GRKKRRQRRRTPQ (SEQ ID NO:8), YGRKKRRQRRR (SEQ ID NO:9), AAVALLPAVLLALLA (SEQ ID NO:10), MGLGLHLLVLAAALQ (SEQ ID NO:11), GALFLGFLGAAGSTM (SEQ ID NO:12), AGYLLGKINLKALAALAKKIL (SEQ ID NO:13), RVIRVWFQNKRCKDKK (SEQ ID NO:14), RQIKFQNRRMKWKK (SEQ ID NO:15), GLFEAIAGFIENGWEGMIDG (SEQ ID NO:16), GWTLNSAGYLLGKIN (SEQ ID NO:17), RSQSRSRYYRQRQRS (SEQ ID NO:18), LAIPEQEY (SEQ ID NO:19), LGIAEQEY (SEQ ID NO:20), LGIPAQAQEY (SEQ ID NO:21), IPAEY (SEQ ID NO:22), IPAGAAY (SEQ ID NO:23), EAIALGELGY (SEQ ID NO:24), IPAAY (SEQ ID NO:25), IAEQAY (SEQ ID NO:26), AZEAY (SEQ ID NO: 4627), SEQ ID NO: 4629 (SEQ ID NO:30), EALAIPEQEYELGEYELGEY (SEQ ID NO:24), and LGAY (SEQ ID NO: 4625), KETWEWTWEWTWWSQPKKKRKV (SEQ ID NO:31), DHQLNPAF (SEQ ID NO:32), DPKGDPKG (SEQ ID NO:33), VTVTVTVTVTGKGDPKPD (SEQ ID NO:34), RQIKIWFQNRRMKWKK (SEQ ID NO:35), GRKKRRQRRPPQ (SEQ ID NO:36), GWTLNSAGYLLGKINLKALAAL AKKIL (SEQ ID NO:37), GRKKRRQRRRR (SEQ ID NO:38), RRRRRRRRRRR (SEQ ID NO:39), RRRRRRRRRRRRRRRRRRRRRRRRRRRR (SEQ ID NO:40), RRRRRRRRR (SEQ ID NO:41), RRRRRR RRRR (SEQ ID NO:42), RRRRRRRRRRR (SEQ ID NO:43) and YTIWMPENPRPGT PCDIFTNSRGKRASNGGG G (R) n, wherein n is 2-15R in L-and/or D form (SEQ ID NO:44), or a cell penetrating fragment thereof.
In some embodiments, the liposome comprises a mitochondrial penetrating agent selected from the group consisting of: guanidine-rich peptoids, tetraguanidines, triguanidines, bis-monoguanidines, guanidine-rich polyurethanes, beta-oligoarginine, proline-rich dendrimers, and phosphonium salts (e.g., methyltriphenylphosphonium and/or tetraphenylphosphonium).
In some embodiments, the liposome comprises sphingomyelin and/or stearyl-octa-arginine. In some embodiments, the liposome comprises sphingomyelin and/or stearyl-octa-arginine. In some embodiments, the liposome comprises DOPE, sphingomyelin, stearyl-octa-arginine sphingomyelin, and stearyl-octa-arginine. In some embodiments, the liposomes comprise DOPE, sphingomyelin, stearyl-octa-arginine sphingomyelin, and stearyl-octa-arginine in a 9:2:1 molar ratio. In some embodiments, the liposome comprises a MITO-porter system or a variant thereof.
In some embodiments, the liposomes comprise an agent, such as a cell penetrating agent, that facilitates delivery of the liposome across the cell membrane and provides the liposome with the ability to bypass the endocytic pathway and the harsh environment of the lysosome. Cell penetrating agents are known in the art and are routinely used and suitable for making and using alpha PMTX compositions. In some embodiments, the cell penetrating/lysosomal bypassing agent is chloroquine. In some embodiments, the cell penetrating agent is a cell penetrating peptide. In some embodiments, the liposome comprises a cell penetrating agent selected from the group consisting of: RQIKIWFQNRRMKWKKRKKRRQR RR (SEQ ID NO:1), RKKRRXR RRGC wherein X is any natural or non-natural amino acid (SEQ ID NO:2), CCGCCAAGAAGCG (SEQ ID NO:3), GCGTGCACACGCGCGTAGACTTCCCCCGCAAGTCACTCGTTAGCCCGC CAAGAAGCGACCCCTCCGGGGCGAGCTGAG CGGCGTGGCGCGGGGGCGTCAT (SEQ ID NO:4), ACGTGCATACGCACGTAGACATTCCCCGCTTCCCACTCCAAA GTCCGCCAAGAAGCGTATCCCGCTGAGCGGCGTGGCGCGGGGGCGTCAT CCGTCAGCTC (SEQ ID NO:5) or ACTTCCCCCGCAAGTCACTCGTTAGCCCGC CAAGAAGCGACCCCTCCGGGGCGAGCTG (SEQ ID NO:6)), or a mitochondrion-penetrating fragment thereof.
In some embodiments, the liposomes in the provided liposome compositions comprise a mitochondrial penetrating agent selected from the group consisting of: guanidine-rich peptoids, tetraguanidines, triguanidines, bis-monoguanidines, guanidine-rich polyurethanes, beta-oligoarginine, proline-rich dendrimers, and phosphonium salts (e.g., methyltriphenylphosphonium and/or tetraphenylphosphonium).
In some embodiments, the liposomes in provided liposome compositions comprise sphingomyelin and/or stearyl-octa-arginine. In some embodiments, the liposome comprises sphingomyelin and/or stearyl-octa-arginine. In some embodiments, the liposome comprises DOPE, sphingomyelin, stearyl-octa-arginine sphingomyelin, and stearyl-octa-arginine. In some embodiments, the liposomes comprise DOPE, sphingomyelin, stearyl-octa-arginine sphingomyelin, and stearyl-octa-arginine in a 9:2:1 molar ratio. In some embodiments, the liposome comprises
Figure BDA0002715283060000501
A system or a variant thereof.
In some embodiments, the liposomes in the provided liposome compositions comprise an agent, such as a cell penetrating agent, that facilitates delivery of the liposome across the cell membrane and provides the liposome with the ability to bypass the endocytic pathway and the harsh environment of the lysosome. Cell penetrating agents are known in the art and are routinely used and suitable for making and using the provided liposome compositions. In some embodiments, the cell penetrating/lysosomal bypassing agent is chloroquine. In some embodiments, the cell penetrating agent is a cell penetrating peptide. In some embodiments, the liposomes in the provided liposome compositions comprise a cell penetrating agent selected from the group consisting of: RKKRRQRRR (SEQ ID NO:7), GRKKRRQRRRTPQ (SEQ ID NO:8), YGRKKRRQRRR (SEQ ID NO:9), AAVAL LPAVLLALLA (SEQ ID NO:10), MGLGLHLLVLAAALQ (SEQ ID NO:11), GALFL GFLGAAGSTM (SEQ ID NO:12), AGYLLGKINLKALAALAKKIL (SEQ ID NO:13), RVIRVWFQNKRCKDKK (SEQ ID NO:14), RQIKFQNRRMKWKK (SEQ ID NO:15), GLFEAIAGFIENGWEGMIDG (SEQ ID NO:16), GWTLNSAGYLLGKIN (SEQ ID NO:17), RSQSRSRYYRQRQRS (SEQ ID NO:18), LAIPEQEY (SEQ ID NO:19), LGIAEQEY (SEQ ID NO:20), LGIPAQAQEY (SEQ ID NO:21), IPAEY (SEQ ID NO:22), IPAQAY (SEQ ID NO:23), EAIALGELGY (SEQ ID NO:24), IPAY (SEQ ID NO:25), SEQ ID NO: EQAY (SEQ ID NO:26), AY LLIILRRRIRKQAHAHSK), EALGEALGEALGEALGELGE (SEQ ID NO:24), EPAY (SEQ ID NO: LLIILRRRIRKQAHAHSK), SEQ ID NO:24, KLALKLALKALKAALKLA (SEQ ID NO:30), KETWWETWWTEWSQPKKKRKV (SEQ ID NO:31), DHQLNPAF (SEQ ID NO:32), DPKGDPKG (SEQ ID NO:33), VTVTVTVTVTGKGDPKPD (SEQ ID NO:34), RQIKIWFQNRRMKWKK (SEQ ID NO:35), GRKKRRQRRPPQ (SEQ ID NO:36), GWTLNSAGYLLGKINLKALAAL AKKIL (SEQ ID NO:37), GRKKRRQRRRR (SEQ ID NO:38), RRRRRRRRRRRRRRR (SEQ ID NO:39), RRRRRRRRRRRRRRRRRRRRRR (SEQ ID NO:40), RRRRRRRRR (SEQ ID NO:41), RRRRRRRR RR (SEQ ID NO:42), RRRRRRRRRRR (SEQ ID NO:43) and YTIWMPENPRPGT PCDIFTNSRGKRASNGGG G (R) n, wherein n is 2-15R in L-and/or D form (SEQ ID NO:44), or a cell penetrating fragment thereof.
As mentioned above, liposomes may contain steric stabilizers, which may extend their circulation life. For those embodiments incorporating a steric stabilizer, the steric stabilizer may be at least one member selected from the group consisting of: polyethylene glycol (PEG); poly-L-lysine (PLL); monosialoganglioside (GM 1); poly (vinyl pyrrolidone) (PVP); poly (acrylamide) (PAA); poly (2-methyl-2-oxazoline); poly (2-ethyl-2-oxazoline); a phosphatidylpolyglycerol; poly [ N- (2-hydroxypropyl) methacrylamide ]; amphiphilic poly-N-vinylpyrrolidone; an L-amino acid-based polymer; oligomerization of glycerol; copolymers comprising polyethylene glycol and polypropylene oxide; poloxamer 188; and polyvinyl alcohol. In some embodiments, the steric stabilizer or population of steric stabilizers is PEG. In one embodiment, the steric stabilizer is PEG. In another embodiment, the PEG has a number average molecular weight (Mn) of 200 to 5000 daltons. These PEGs may have any structure, such as linear, branched, star-shaped, or comb-shaped structures, and are commercially available.
In some embodiments, the liposome composition comprises pegylated liposomes (PLp- α PMTX). In some embodiments, the pegylated liposomes in the liposome compositions comprise alpha PMTX containing 4, 5, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the alpha polyglutamated methotrexate in the Lp-alpha PMTX comprises two or more glutamyl groups in the L form. In other embodiments, the α polyglutamated methotrexate in Lp- α PMTX comprises a glutamyl group in the D form. In other embodiments, the α polyglutamated methotrexate in the Lp- α PMTX comprises a glutamyl group in the D form and two or more glutamyl groups in the L form. In further embodiments, the α polyglutamated methotrexate in the Lp- α PMTX comprises two or more glutamyl groups with γ linkages. In some embodiments, at least one glutamyl group has both an alpha linkage and a gamma linkage. In some embodiments, the liposome composition comprises a pegylated liposome comprising alpha pentaglutamated MTX. In other embodiments, the liposome comprises L-alpha pentaglutamated MTX, D-alpha pentaglutamated MTX, or L-and D-alpha pentaglutamated MTX. In some embodiments, the liposome composition comprises a pegylated liposome comprising alpha hexaglutaminated MTX. In other embodiments, the liposome comprises L-alpha hexaglutamated MTX, D-alpha hexaglutamated MTX, or L-and D-alpha hexaglutamated MTX. In some embodiments, the liposome composition comprises an anionic or neutral pegylated liposome. In some embodiments, the liposome composition comprises a cationic pegylated liposome. In some embodiments, the PLp- α PMTX composition is non-targeted (NTPLp- α PMTX). In other embodiments, the PLp- α PMTX composition is targeted (TPLp- α PMTX). In further embodiments, the liposome composition comprises pegylated liposomes comprising 30% -70%, 30% -60%, or 30% -50% liposome-entrapped alpha polyglutamated methotrexate, or any range therebetween. In some embodiments, at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75% (w/w) of alpha polyglutamated methotrexate is encapsulated (embedded) in PLp-alpha PMTX. In some embodiments, the liposome composition comprises pegylated liposomes having a diameter in the range of 20nm to 500 nm. In some embodiments, the liposome composition comprises pegylated liposomes having a diameter in the range of 20nm to 200 nm. In other embodiments, the liposome composition comprises pegylated liposomes having a diameter in the range of 80nm to 120 nm.
In some embodiments, greater than 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the polyglutamated methotrexate in the composition has 4-10, 4-6, or more than 5 glutamyl groups. In some embodiments, provided liposome compositions have greater than 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the polyglutamated methotrexate is tetraglutamated. In some embodiments, provided liposome compositions have greater than 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the polyglutamated methotrexate is pentaglutamated. In some embodiments, provided liposome compositions have greater than 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the polyglutamated methotrexate is hexaglutamated.
In some embodiments, the alpha polyglutamated methotrexate composition (e.g., polyglutamate and a delivery vehicle, such as a liposome containing polyglutamate) is in an aqueous solution. In some embodiments, in terms of per square meter (m)2) Administering a dose of alpha PMTX between 0.005 and 5000mg of body surface area, or any range therebetween, in the form of a liposome composition. In other embodiments, the alpha PMTX composition is administered in the form of a liposome composition at a dose of between 0.1 and 1000mg of alpha PMTX per square meter of body surface area, or any range therebetween.
(1) Liposome composition
The lipids and other components of the liposomes contained in the liposome composition may be any lipids, the combination and proportions of lipids or the combination of lipids and other liposome components and their respective proportions are known in the art. However, one skilled in the art will appreciate that liposomal encapsulation of any particular drug, such as, but not limited to, the α polyglutamated MTX discussed herein, can involve a number of routine experiments to achieve useful and functional liposomal formulations. In general, the provided liposomes can have any liposomal structure, e.g., a structure having an interior space isolated from an external medium by one or more lipid bilayers, or any microcapsule having a semi-permeable membrane with a lipophilic central portion, wherein the membrane isolates the interior. The lipid bilayer may be any arrangement of amphipathic molecules characterized by hydrophilic portions (hydrophilic portions) and hydrophobic portions (hydrophobic portions). In general, the amphipathic molecules in the bilayer are arranged in a two dimensional sheet with the hydrophobic portions oriented inward and the hydrophilic portions oriented outward. The amphipathic molecules forming the provided liposomes can be any known or later discovered amphipathic molecules, such as synthetic or naturally derived lipids or biocompatible lipids. Liposomes can also be formed from amphiphilic polymers and surfactants, for example, polymeric bodies and vesicles. For the purposes of this disclosure, but not by way of limitation, these liposome-forming materials are also referred to as "lipids".
The liposome composition formulations provided herein can be in liquid or dry form, such as a dry powder or a dry cake. The dry powder or dry cake can be subjected to primary drying, for example, under lyophilization conditions, or optionally, the dry cake or dry powder can be subjected to only primary drying or both primary and secondary drying. In dry form, the powder or filter cake may for example have a moisture of 1% to 6%, for example between 2% to 5% or between 2% to 4%. An exemplary method of drying is lyophilization (also known as freeze-drying or lyophilization). Any of the compositions and methods of the present disclosure can include liposomes, lyophilized liposomes, or liposomes reconstituted from lyophilized liposomes. In some embodiments, the disclosed compositions and methods include one or more lyoprotectants or cryoprotectants. These protective agents are generally polyhydroxy compounds, such as sugars (mono-, di-and polysaccharides), polyols and their derivatives, glycerol or polyethylene glycols, trehalose, maltose, sucrose, glucose, lactose, dextran, glycerol or aminoglycosides. In other embodiments, the lyoprotectant or cryoprotectant comprises up to 10% or up to 20% of the solution outside the liposome, inside the liposome, or both outside and inside the liposome.
In some embodiments, the liposomes include a steric stabilizer that extends their lifetime in circulation. One or more steric stabilizers such as hydrophilic polymers (polyethylene glycol (PEG)), glycolipids (monosialoganglioside (GM1)) or other substances occupy the space immediately adjacent to the liposome surface and exclude other macromolecules from this space. Thus, access to and binding of plasma opsonin to the liposome surface is hindered, and thus interaction of macrophages with such liposomes or any other clearance mechanism is inhibited, and the lifetime of the liposomes in circulation is enhanced. In some embodiments, the steric stabilizer or population of steric stabilizers is PEG or a combination comprising PEG. In other embodiments, the steric stabilizer is PEG or a combination comprising PEG having a number average molecular weight (Mn) of 200 to 5000 daltons. These PEGs may have any structure, such as linear, branched, star-shaped, or comb-shaped structures, and are commercially available.
The diameter of the disclosed liposomes is not particularly limited. In some embodiments, the liposomes have a diameter in the range of, for example, 30-150nm (nanometers). In other embodiments, the liposomes have a diameter in the range of 40-70 nm.
The properties of liposomes are influenced by the properties of the lipids used to prepare the liposomes. A wide variety of lipids have been used to prepare liposomes. These include cationic lipids, anionic lipids and neutral lipids. In some embodiments, the liposome comprising alpha-polyglutamated methotrexate is anionic or neutral. In other embodiments, provided liposomes are cationic. The charge (e.g., anionic, neutral, or cationic) can be determined conventionally by measuring the zeta potential of the liposome. The zeta potential of the liposomes can be positive, zero, or negative. In some embodiments, the zeta potential of the liposome is less than or equal to zero. In some embodiments, the zeta potential of the liposome is in the range of 0 to-150 mV. In another embodiment, the zeta potential of the liposomes is in the range of-30 to-50 mV.
In some embodiments, cationic lipids are used to prepare cationic liposomes, which are typically used as gene transfection agents. The positive charge on the cationic liposome can interact with the negative charge on the cell surface. After the cationic liposome is bound to the cell, the liposome is transported within the cell by endocytosis.
In some preferred embodiments, neutral to anionic liposomes are used. In a preferred embodiment, anionic liposomes are used. The use of a mixture of, for example, neutral lipids such as HSPC and anionic lipids such as PEG-DSPE, allows the formation of anionic liposomes that are less likely to bind non-specifically to normal cells. Specific binding to tumor cells can be achieved by using tumor targeting antibodies, such as folate receptor antibodies, including, for example, folate receptor alpha antibodies, folate receptor beta antibodies, and/or folate receptor antibodies.
As an example, at least one (or some) lipid is an amphiphilic lipid, defined as having a hydrophilic portion and a hydrophobic portion (typically a hydrophilic head and a hydrophobic tail). The hydrophobic portion is generally oriented as the hydrophobic phase (e.g., within the bilayer), while the hydrophilic portion is generally oriented as the aqueous phase (e.g., outside the bilayer). The hydrophilic moiety may comprise polar or charged groups such as carbohydrates, phosphates, carboxylic acids, sulfates, amino groups, sulfhydryls, nitro groups, hydroxyl groups, and other similar groups. The hydrophobic moiety may comprise a non-polar group including, but not limited to, long chain saturated and unsaturated aliphatic hydrocarbon groups and groups substituted with one or more aromatic, alicyclic, or heterocyclic groups. Examples of amphiphilic compounds include, but are not limited to, phospholipids, amino lipids, and sphingolipids.
Typically, for example, the lipid is a phospholipid. Phospholipids include, but are not limited to, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, and the like. It will be appreciated that other lipid membrane components may be used, such as cholesterol, sphingomyelin and cardiolipin.
The lipids comprising the liposomes provided herein can be anionic and neutral (including zwitterionic and polar) lipids, including anionic and neutral phospholipids. At a selected pH, neutral lipids exist in the uncharged or neutral zwitterionic form. At physiological pH, such lipids include, for example, Dioleoylphosphatidylglycerol (DOPG), diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, cerebrosides, and diacylglycerol. Examples of zwitterionic lipids include, but are not limited to, Dioleoylphosphatidylcholine (DOPC), Dimyristoylphosphatidylcholine (DMPC), and Dioleoylphosphatidylserine (DOPS). Anionic lipids are negatively charged at physiological pH. These lipids include, but are not limited to, phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanolamine, N-succinylphosphatidylethanolamine, N-glutarylphosphatidylethanolamine, lysylphosphatidylglycerol, palmitoyloleoyl-phosphatidylglycerol (POPG), and other anionic modifying groups attached to neutral lipids.
Collectively, anionic lipids and neutral lipids are referred to herein as non-cationic lipids. Such lipids may contain phosphorus, but are not limited thereto. Examples of non-cationic lipids include lecithin, lysolecithin, phosphatidylethanolamine, lysophosphatidylethanolamine, Dioleoylphosphatidylethanolamine (DOPE), Dipalmitoylphosphatidylethanolamine (DPPE), Dimyristoylphosphatidylethanolamine (DMPE), distearoylphosphatidyl1-ethanolamine (DSPE), palmitoylphosphatidylethanolamine (POPE), palmitoyl-oleoylphosphatidylcholine (POPC), Egg Phosphatidylcholine (EPC), Distearoylphosphatidylcholine (DSPC), Dioleoylphosphatidylcholine (DOPC), Dipalmitoylphosphatidylglycerol (DPPC), Dioleoylphosphatidylglycerol (DOPG), Dipalmitoylphosphatidylglycerol (DPPG), palmitoylphosphatidylglycerol (POPG), 16-0-methyl PE, 16-0-dimethyl PE, 18-1-trans PE, palmitoyl oleoyl-phosphatidylethanolamine (POPE), 1-stearoyl-2-oleoyl phosphatidylethanolamine (SOPE), phosphatidylserine, phosphatidylinositol, sphingomyelin, cephalin, cardiolipin, phosphatidic acid, cerebroside, dicetyl phosphate, and cholesterol.
Liposomes can be assembled using any liposome assembly method known in the art that uses a liposome component (also known as a liposome component). Liposomal components include, for example, lipids such as DSPE, HSPC, cholesterol, and derivatives of these components. Other suitable Lipids are commercially available, for example, from Avanti Polar Lipids, Inc (Alabaster, Alabama, USA). A partial list of useful negatively or neutrally charged lipids suitable for preparing anionic liposomes can be, for example, at least one of: DLPC, DMPC, DPPC, DSPC, DOPC, DMPE, DPPE, DOPE, DMPA. Na, DPPA. Na, DOPA. Na, DMPG. Na, DPPG. Na, DOPG. Na, DMPS. Na, DPPS. Na, DOPS. Na, DOPE-glutaryl (Na)2, tetramyristoyl cardiolipin (Na)2, DSPE-mPEG-2000. Na, DSPE-mPEG-5000. Na, and DSPE-maleimide PEG-2000. Na.
In some embodiments, the α PMTX compositions provided herein are formulated in liposomes comprising cationic lipids. In one embodiment, the cationic lipid is selected from, but is not limited to, the cationic lipids described in international application publication nos. WO2012/040184, WO2011/153120, WO2011/149733, WO2011/090965, WO2011/043913, WO2011/022460, WO2012/061259, WO2012/054365, WO2012/044638, WO2010/080724, WO2010/21865, and WO2008/103276, U.S. patent nos. 7,893,302, 7,404,969, and 8,283,333, and U.S. application publication nos. US20100036115 and US20120202871, each of which is incorporated herein by reference in its entirety. In another embodiment, the cationic lipid may be selected from, but is not limited to, formula a described in international application publication nos. WO2012/040184, WO2011/153120, WO201/1149733, WO2011/090965, WO2011/043913, WO2011/022460, WO2012/061259, WO2012/054365, and WO2012/044638, each of which is incorporated herein by reference in its entirety. In another embodiment, the cationic lipid may be selected from, but is not limited to, those of formula CLI-CLXXIX of international publication No. WO2008103276, CLI-CLXXIX of U.S. patent No. 7,893,302, CLI-clxxxii of U.S. patent No. 7,404,969, and formula I-VI of U.S. patent publication No. US 20100036115; each of which is incorporated herein by reference in its entirety. As a non-limiting example, the cationic lipid may be selected from (20Z,23Z) -N, N-dimethylmontan-20, 23-dien-10-amine, (17Z,20Z) -N, N-dimethyl-hexacosan-17, 20-dien-9-amine, (1Z,19Z) -N5N-dimethylpentacosan-16, 19-dien-8-amine, (13Z,16Z) -N, N-dimethyldocosan-13, 16-dien-5-amine, (12Z,15Z) -N, N-dimethylheneicosan-12, 15-dien-4-amine, (14Z,17Z) -N, N-dimethyltricosan-14, 17-dien-6-amine, (15Z,18Z) -N, N-dimethylditetradec-15, 18-dien-7-amine, (18Z,21Z) -N, N-dimethylheptacosan-18, 21-dien-10-amine, (15Z,18Z) -N, N-dimethylditetradec-15, 18-dien-5-amine, (14Z,17Z) -N, N-dimethyl-tricosane-14, 17-dien-4-amine, (19Z,22Z) -N, N-dimethyldioctadecyl-19, 22-dien-9-amine, (18Z,21Z) -N, N-dimethylheptacosa-18, 21-dien-8-amine, (17Z,20Z) -N, N-dimethylhexacosan-17, 20-dien-7-amine, (16Z,19Z) -N, N-dimethylpentacosan-16, 19-dien-6-amine, (22Z,25Z) -N, N-dimethylhentriacontan-22, 25-dien-10-amine, (21Z,24Z) -N, N-dimethyl-triaconta-21, 24-dien-9-amine, (18Z) -N, N-dimethylheptacosan-18-en-10-amine, (17Z) -N, N-dimethylhexacosan-17-en-9-amine, (19Z,22Z) -N, N-dimethyldioctadecyl-19, 22-dien-7-amine, N-dimethylheptacosan-10-amine, (20Z,23Z) -N-ethyl-N-methylnonacosan-20, 23-dien-10-amine, 1- [ (11Z,14Z) -1-nonyleicosa-11, 14-dien-1-yl ] pyrrolidine, (20Z) -N, N-dimethylheptacosan-20-en-10-amine, (15Z) -N, N-dimethylheptacosan-15-en-10-amine, (14Z) -N, N-dimethylnonacosan-14-en-10-amine, (17Z) -N, N-dimethylnonacosan-17-en-10-amine, and mixtures thereof, (24Z) -N, N-dimethyltridec-24-en-10-ylamine, (20Z) -N, N-dimethylmontan-20-en-10-ylamine, (22Z) -N, N-dimethylhentriacont-22-en-10-ylamine, (16Z) -N, N-dimethylpentacosan-16-en-8-ylamine, (12Z,15Z) -N, N-dimethyl-2-nonylheneicosyl-12, 15-dien-1-ylamine, (13Z,16Z) -N, N-dimethyl-3-nonyldidodeca-13, 16-dien-1-ylamine, N-dimethyl-1- [ (1S,2R) -2-octylcyclopropyl ] heptadec-8-amine, 1- [ (1S,2R) -2-hexylcyclopropyl ] -N, N-dimethylnona-10-amine, N-dimethyl-1- [ (1S,2R) -2-octylcyclopropyl ] nonadeca-10-amine, N-dimethyl-21- [ R1S,2R) -2-octylcyclopropyl ] heneico-10-amine, N-dimethyl-1- [ (1S,2S) -2- { [ (1R,2R) -2-pentylcyclopropyl ] methyl } cyclopropyl ] nona-10-amine, N-dimethyl-1- [ (1S,2R) -2-octylcyclopropyl ] hexadec-8-amine, N-dimethyl- [ (1R,2S) -2-undecyl-cyclopropyl ] tetradec-5-amine, N-dimethyl-3- {7- [ (1S,2R) -2-octylcyclopropyl ] heptyl } dodeca-1-amine, 1- [ (1R,2S) -2-heptylcyclopropyl ] -N, N-dimethyloctadeca-9-amine, 1- [ (1S,2R) -2-decylcyclopropyl ] -N, N-dimethylpentadec-6-amine, N-dimethyl-1- [ (1S,2R) -2-octylcyclopropyl ] pentadecan-8-amine, N-dimethylcy-1-ylpropyl ] -N, N-dimethylpentadecan-6-amine, N-dimethylcy-1- [ (1S,2R) -2-octylcyclopropyl, R-N, N-dimethyl-1- [ (9Z,12Z) -octadec-9, 12-dien-1-yloxy ] -3- (octyloxy) propan-2-amine, S-N, N-dimethyl-1- [ (9Z,12Z) -octadec-9, 12-dien-1-yloxy ] -3- (octyloxy) propan-2-amine, 1- {2- [ (9Z,12Z) -octadec-9, 12-dien-1-yloxy ] -1- [ (octyloxy) methyl ] ethyl } pyrrolidine, (2S) -N, N-dimethyl-1- [ (9Z,12Z) -octadec-9, 12-dien-1-yloxy ] -3- [ (5Z) -oct-5-en-1-yloxy ] propan-2-amine, 1- {2- [ (9Z,12Z) -octadec-9, 12-dien-1-yloxy ] -1- [ (octyloxy) methyl ] ethyl } azetidine, (2S) -1- (hexyloxy) -N, N-dimethyl-3- [ (9Z,12Z) -octadec-9, 12-dien-1-yloxy ] propan-2-amine, (2S) -1- (heptyloxy) -N, N-dimethyl-3- [ (9Z,12Z) -octadec-9, 12-dien-1-yloxy ] propan-2-amine, N-dimethyl-1- (nonyloxy) -3- [ (9Z,12Z) -octadec-9, 12-dien-1-yloxy ] propan-2-amine, N-dimethyl-1- [ (9Z) -octadec-9-en-1-yloxy ] -3- (octyloxy) propan-2-amine; (2S) -N, N-dimethyl-1- [ (6Z,9Z,12Z) -octadeca-6, 9, 12-trien-1-yloxy ] -3- (octyloxy) propan-2-amine, (2S) -1- [ (11Z,14Z) -eicosa-11, 14-dien-1-yloxy ] -N, N-dimethyl-3- (pentyloxy) propan-2-amine, (2S) -1- (hexyloxy) -3- [ (11Z,14Z) -eicosa-11, 14-dien-1-yloxy ] -N, N-dimethylpropan-2-amine, 1- [ (11Z,14Z) -eicosa-11, 14-dien-1-yloxy ] -N, N-dimethyl-3- (octyloxy) propan-2-amine, 1- [ (13Z,16Z) -docosan-13, 16-dien-1-yloxy ] -N, N-dimethyl-3- (octyloxy) propan-2-amine, (2S) -1- [ (13Z,16Z) -docosan-13, 16-dien-1-yloxy ] -3- (hexyloxy) -N, N-dimethylpropan-2-amine, (2S) -1- [ (13Z) -docosan-13-en-1-yloxy ] -3- (hexyloxy) -N, n-dimethylprop-2-amine, 1- [ (13Z) -docosan-13-en-1-yloxy ] -N, N-dimethyl-3- (octyloxy) propan-2-amine, 1- [ (9Z) -hexadec-9-en-1-yloxy ] -N, N-dimethyl-3- (octyloxy) propan-2-amine, (2R) -N, N-dimethyl-H (1-formyloctyl) oxy ] -3- [ (9Z,12Z) -octadeca-9, 12-dien-1-yloxy ] propan-2-amine, (2R) -1- [ (3, 7-dimethyloctyl) oxy ] -N, N-dimethyl-3-R9Z, 12Z) -octadeca-9, 12-dien-1-yloxy ] propan-2-amine, N-dimethyl-1- (octyloxy) -3- ({8- [ (1S,2S) -2- { [ (1R,2R) -2-pentylcyclopropyl ] methyl } cyclopropyl ] octyl } oxy) propan-2-amine, n, N-dimethyl-1- { [ - (2-octylcyclopropyl) octyl ] oxy } -3- (octyloxy) propan-2-amine and (11E,20Z,23Z) -N, N-dimethyl-nonacosane-11, 20, 2-trien-10-amine or a pharmaceutically acceptable salt or acid stereoisomer thereof.
In one embodiment, the lipid may be a cleavable lipid, such as those described in international publication No. WO2012/170889, which is incorporated herein by reference in its entirety.
Cationic lipids can be conventionally synthesized using methods known in the art and/or as described in international publication nos. WO2012/040184, WO2011/153120, WO2011/149733, WO2011/090965, WO201/1043913, WO2011/022460, WO2012/061259, WO2012/054365, WO2012/044638, WO2010/080724, and WO2010/21865, each of which is incorporated herein by reference in its entirety.
The lipid derivative may comprise, for example, at least a bonding (preferably covalent bonding) of one or more steric stabilizers and/or functional groups to the liposomal composition, after which the steric stabilizers and/or functional groups should be considered as part of the liposomal composition. Functional groups include groups that can be used to attach a liposome component to another moiety, such as a protein. Such functional groups include at least maleimide. These steric stabilizers include at least one selected from the group consisting of: polyethylene glycol (PEG); poly-L-lysine (PLL); monosialoganglioside (GM 1); poly (vinyl pyrrolidone) (PVP); poly (acrylamide) (PAA); poly (2-methyl-2-oxazoline); poly (2-ethyl-2-oxazoline); a phosphatidylpolyglycerol; poly [ N- (2-hydroxy-propyl) methacrylamide ]; amphiphilic poly-N-vinylpyrrolidone; an L-amino acid based polymer; and polyvinyl alcohol.
In some embodiments, the alpha PMTX composition is formulated in a lipid-polycation complex. Formation of the lipid-polycation complex may be accomplished using methods known in the art and/or as described in U.S. publication No. 20120178702, which is incorporated herein by reference in its entirety. As non-limiting examples, the polycation may include cationic peptides or polypeptides such as, but not limited to, polylysine, polyornithine, and/or polyarginine and cationic peptides described in international publication No. WO 2012/013326; the publication is incorporated by reference herein in its entirety. In another embodiment, the α PMTX is formulated in a lipid-polycation complex that further comprises a neutral lipid, such as, but not limited to, cholesterol or Dioleoylphosphatidylethanolamine (DOPE).
As the components of the liposomes can include any molecule (i.e., chemical/agent/protein) bound thereto, in some embodiments, the components of the provided liposomes include at least a member selected from the group consisting of: DSPE, DSPE-PEG, DSPE-maleimide, HSPC; HSPC-PEG; HSPC-maleimide; cholesterol; cholesterol-PEG; and cholesterol-maleimide. In some embodiments, provided components of liposomes include DSPE, DSPE-PEG, DSPE-maleimide, HSPC; HSPC-PEG; HSPC maleimides; cholesterol; cholesterol-PEG; and cholesterol-maleimide. In a preferred embodiment, the liposome components making up the liposomes comprise DSPE; DSPE-FITC; DSPE-maleimide; cholesterol; and HSPC.
In further embodiments, the liposomes of the liposome compositions provided herein comprise an oxidized phospholipid. In some embodiments, the liposome comprises an oxidized phospholipid selected from the group consisting of: phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine, phosphatidylcholine, and 1-palmitoyl-2-arachidonoyl-sn-glycero-2-phosphate. In some embodiments, the phospholipid has an unsaturated bond. In some embodiments, the phospholipid is an arachidonic acid-containing phospholipid. In further embodiments, the phospholipid is sn-2-oxygenated. In other embodiments, the phospholipid is not fragmented.
In some embodiments, the liposomes of the disclosed liposome compositions comprise oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (oxpac). As used herein, the term "oxPAPC" refers to lipids produced by oxidation of 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine (PAPC) resulting in a mixture of oxidized phospholipids containing fragmented or full length oxygenated sn-2 residues. Well characterized oxidatively fragmented species contain five carbon sn-2 residues carrying either an omega-aldehyde or an omega-carboxyl group. Oxidation of arachidonic acid residues also produces phospholipids containing esterified isoprostane. OxPAPC includes the classes HOdia-PC, KOdia-PC, HOOA-PC and KOOA-PC, in addition to other oxidation products present in the oxPAPC. In other embodiments, the oxPAPC is an epoprostane-containing phospholipid. In other embodiments, the oxPAPC is 1-palmitoyl-2- (5, 6-epoxyisoprostane E2) -sn-glycero-3-phosphocholine (5,6-PEIPC), 1-palmitoyl-2- (epoxy-cyclopentenone) -sn-glycero-3-phosphorylcholine (PECPC) and/or 1-palmitoyl-2- (epoxy-isoprostane E2) -sn-glycero-4-phosphocholine (PEIPC). In some embodiments, the phospholipid has an unsaturated bond. In some embodiments, the phospholipid is an arachidonic acid-containing phospholipid. In further embodiments, the phospholipid is sn-2-oxygenated. In other embodiments, the phospholipid is not fragmented.
In some embodiments, the liposomal α -polyglutamated methotrexate composition is pegylated (i.e., pegylated liposomal α -polyglutamated (e.g., pentaglutamated or hexaglutamated) antifolate (PLp- α PMTX or PLp- α PMTX)). In some embodiments, the PLp- α PMTX or PLp- α PMTX is water soluble. That is, PLp- α PMTX or PLp- α PMTX is in the form of an aqueous solution.
In some embodiments, the liposomes of the disclosed liposome compositions comprise a lipid selected from the group consisting of: 1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine (PGPC); 1-palmitoyl-2- (9' oxo-nonanoyl) -sn-glycero-3-phosphocholine; 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine; 1-palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine; 1-palmitoyl-2-hexadecyl-sn-glycero-3-phosphocholine; 1-palmitoyl-2-nonanedioyl-sn-glycero-3-phosphocholine; and 1-palmitoyl-2-acetyl-sn-glycero-3-phosphocholine. In other embodiments, the liposome comprises PGPC.
In some embodiments, the pH of the solution comprising the liposome composition is pH 2 to 8, or any range therebetween. In some embodiments, the pH of the solution comprising the liposome composition is pH 5 to 8, or any range therebetween.
In some embodiments, at least one component of the liposomal lipid bilayer is functionalized (or reactive). As used herein, a functionalized component is a component that comprises reactive groups that can be used to crosslink reagents and moieties to lipids. If the lipid is functionalized, any liposomes it forms are also functionalized. In some embodiments, the reactive group is a group that will react with the crosslinker (or other moiety) to form a crosslink. The reactive group in the liposomal lipid bilayer is located anywhere on the lipid that allows it to contact the cross-linking agent and cross-link with another moiety (e.g., a steric stabilizer or targeting moiety). In some embodiments, the reactive group is in the head group of the lipid, including, for example, phospholipids. In some embodiments, the reactive group is a maleimide group. The maleimide groups may be crosslinked to each other in the presence of dithiol crosslinkers, including but not limited to Dithiothreitol (DTT).
It is to be understood that the use of other functionalized lipids, other reactive groups, and other cross-linking agents than those described above are further contemplated. In addition to maleimide groups, other examples of reactive groups contemplated include, but are not limited to, other thiol-reactive groups, amino groups such as primary and secondary amines, carboxyl groups, hydroxyl groups, aldehyde groups, alkynyl groups, azido groups, carbonyl groups, haloacetyl groups (e.g., iodoacetyl groups), imidate groups, N-hydroxysuccinimide esters, sulfhydryl groups, and pyridyl disulfide groups.
Functionalized and non-functionalized Lipids are available from a number of commercial sources, including Avanti Polar Lipids (Alabaster, AL) and Lipoid LLC (Newark, NJ).
(2) Liposome interior space
In other non-limiting embodiments, provided liposomes enclose an interior space. In some embodiments, the interior space includes, but is not limited to, an aqueous solution. In some embodiments, the interior space comprises alpha-polyglutamated methotrexate as provided herein. In further embodiments, the interior space of the liposome comprises a tonicity agent. In some embodiments. In some embodiments, the concentration (weight percent) of the tonicity agent is 0.1% to 20%, 1% to 20%, 0.5% to 15%, 1% to 15%, or 1% to 50%, or any range therebetween. In some embodiments, the interior space of the liposome comprises a sugar (e.g., trehalose, maltose, sucrose, lactose, mannose, mannitol, glycerol, dextrose, fructose, etc.). In other embodiments, the concentration (weight percent) of the sugar is 0.1% -20%, 1% -20%, 0.5% -15%, 1% -15%, or 1% -50%, or any range therebetween. In some embodiments, the pH of the liposome interior space is pH 2 to 8, or any range therebetween. In some embodiments, the pH of the solution comprising the liposome composition is pH 5 to 8, or any range therebetween. In some embodiments, the pH of the solution comprising the liposome composition is pH 6 to 7, or any range therebetween. In some embodiments, the pH of the solution comprising the liposome composition is 6 to 7.5, 6.5 to 7.5, 6.7 to 7.5, or 6.3 to 7.0, or any range therebetween. In some embodiments, the interior space comprises a buffer. In other embodiments, the buffer is a buffer selected from HEPES, citrate, or sodium phosphate (e.g., sodium dihydrogen phosphate and/or disodium hydrogen phosphate). In some embodiments, the buffer is HEPES. In some embodiments, the buffer is citrate. In some embodiments, the buffer is sodium phosphate (e.g., sodium dihydrogen phosphate and/or disodium hydrogen phosphate). In some embodiments, the concentration of the buffer is 15 to 200mM, or any range therebetween. In other embodiments, the buffer has a concentration between 5 to 200mM, 15 to 200, between 5 to 100mM, between 15 to 100mM, between 5 to 50mM, between 15 to 50mM, between 5 to 25mM, between 5 to 20mM, between 5 to 15mM, or any range therebetween. In some embodiments, the buffer is HEPES at a concentration of 15 to 200mM or any range therebetween. In some embodiments, the buffer is citrate at a concentration of 15 to 200mM or any range therebetween. In some embodiments, the buffer is sodium phosphate at a concentration of 15 to 200mM or any range therebetween. In some embodiments, the interior space of the liposome comprises sodium acetate and calcium acetate at a total concentration between 5mM to 500mM, or 50mM to 500mM, or any range therebetween.
In some embodiments, the interior space of the liposome comprises trehalose. In other embodiments, the concentration of trehalose is 0.1% -20%, 1% -20%, 0.5% -15%, 1% -15%, 5% -20%, or 1% -50% by weight, or any range therebetween. In other embodiments, the concentration (weight percent) of trehalose is 1% -15% or any range therebetween. In another embodiment, trehalose is present at about 5% to 20% weight percent of trehalose or any combination of one or more lyoprotectants or cryoprotectants, at a total concentration of 5% to 20%. In some embodiments, the pH of the solution comprising the liposome composition is 6 to 7.5, 6.5 to 7.5, 6.7 to 7.5, or 6.3 to 7.0, or any range therebetween. In some embodiments, the interior space comprises a buffer. In some embodiments, the buffer is selected from HEPES, citrate, or sodium phosphate (e.g., sodium dihydrogen phosphate and/or disodium hydrogen phosphate). In some embodiments, the buffer is HEPES. In some embodiments, the buffer is citrate. In some embodiments, the buffer is sodium phosphate (e.g., sodium dihydrogen phosphate and/or disodium hydrogen phosphate). In some embodiments, the concentration of the buffer is 15 to 200mM, or any range therebetween. In other embodiments, the concentration of HBS citrate buffer is between 5 to 200mM, 15 to 200, between 5 to 100mM, between 15 to 100mM, between 5 to 50mM, between 15 to 50mM, between 5 to 25mM, between 5 to 20mM, between 5 to 15mM, or any range therebetween. In some embodiments, the buffer is HEPES at a concentration of 15 to 200mM or any range therebetween. In some embodiments, the buffer is citrate at a concentration of 15 to 200mM or any range therebetween. In some embodiments, the buffer is sodium phosphate at a concentration of 15 to 200mM or any range therebetween. In further embodiments, the interior space of the liposome comprises sodium acetate and/or calcium acetate. In some embodiments, the interior space of the liposome comprises sodium acetate and calcium acetate at a total concentration between 5mM to 500mM, or 50mM to 500mM, or any range therebetween.
In some embodiments, the interior space of the liposome comprises dextrose. In other embodiments, the concentration of dextrose is 0.1% -20%, 1% -20%, 0.5% -15%, 1% -15%, 5% -20%, or 1% -50% by weight, or any range therebetween. In other embodiments, the concentration (weight percent) of dextrose is 1% -15% or any range therebetween. In another embodiment, dextrose is present at about 5% to 20% weight percent of dextrose or one or more lyoprotectants or any combination of cryoprotectants, for a total concentration of 5% to 20%. In some embodiments, the pH of the solution comprising the liposome composition is 6 to 7.5, 6.5 to 7.5, 6.7 to 7.5, or 6.3 to 7.0, or any range therebetween. In some embodiments, the interior space comprises a buffer. In some embodiments, the buffer is selected from HEPES, citrate, or sodium phosphate (e.g., sodium dihydrogen phosphate and/or disodium hydrogen phosphate). In some embodiments, the buffer is HEPES. In some embodiments, the buffer is citrate. In some embodiments, the buffer is sodium phosphate (e.g., sodium dihydrogen phosphate and/or disodium hydrogen phosphate). In some embodiments, the concentration of the buffer is 15 to 200mM, or any range therebetween. In other embodiments, the buffer has a concentration between 5 to 200mM, 15 to 200, between 5 to 100mM, between 15 to 100mM, between 5 to 50mM, between 15 to 50mM, between 5 to 25mM, between 5 to 20mM, between 5 to 15mM, or any range therebetween. In some embodiments, the buffer is HEPES at a concentration of 15 to 200mM or any range therebetween. In some embodiments, the buffer is citrate at a concentration of 15 to 200mM or any range therebetween. In some embodiments, the buffer is sodium phosphate at a concentration of 15 to 200mM or any range therebetween. In further embodiments, the interior space of the liposome comprises sodium acetate and/or calcium acetate. In some embodiments, the interior space of the liposome comprises sodium acetate and calcium acetate at a total concentration between 5mM to 500mM, or 50mM to 500mM, or any range therebetween.
In additional embodiments, the present disclosure provides liposome compositions comprising liposomes encapsulating (i.e., filled with) alpha polyglutamated methotrexate (e.g., alpha PMTX disclosed herein). In some embodiments, the liposomes in the liposome composition comprise alpha PMTX containing 4, 5, 2-10, 4-6, or more than 5 glutamyl groups (including glutamyl groups in methotrexate). In some embodiments, the alpha polyglutamated methotrexate in the Lp-alpha PMTX comprises two or more glutamyl groups in the L form. In other embodiments, the α polyglutamated methotrexate in Lp- α PMTX comprises a glutamyl group in the D form. In other embodiments, the α polyglutamated methotrexate in the Lp- α PMTX comprises a glutamyl group in the D form and two or more glutamyl groups in the L form. In further embodiments, the α polyglutamated methotrexate in the Lp- α PMTX comprises two or more glutamyl groups with a γ carboxyl linkage. In some embodiments, the liposome composition comprises liposomes comprising alpha pentaglutamated MTX. In other embodiments, the liposome comprises L-alpha pentaglutamated MTX, D-alpha pentaglutamated MTX, or L-and D-alpha pentaglutamated MTX. In some embodiments, the liposome composition comprises liposomes comprising alpha hexaglutaminated MTX (Lp-alpha PMTX). In other embodiments, the liposome comprises L-alpha hexaglutamated MTX, D-alpha hexaglutamated MTX, or L-and D-alpha hexaglutamated MTX.
In some embodiments, the targeted pegylated liposomal alpha polyglutamated (e.g., penta-or hexa-glutamated) methotrexate comprises a medium comprising liposomes comprising an interior space; an aqueous solution of alpha-polyglutamated methotrexate disposed within the interior space; and a targeting moiety comprising a protein having specific affinity for at least one folate receptor, and wherein the targeting moiety is disposed on the exterior of the liposome. In some embodiments, the medium is an aqueous solution. In some embodiments, the interior space, the exterior space (e.g., medium), or both the interior space and the medium contain one or more of the above-listed lyoprotectants or cryoprotectants. In some embodiments, the cryoprotectant is mannitol, trehalose, sorbitol, or sucrose.
In some embodiments, the liposomes encapsulating alpha polyglutamated methotrexate (e.g., Lp-alpha PMTX, including PLp-alpha PMTX, TPLp-alpha PMTX, TLp-alpha PMTX, and NTLp-alpha PMTX) have an interior space containing less than 500,000 or less than 200,000 molecules of alpha polyglutamated methotrexate. In some embodiments, the liposome interior space contains between 10 and 100,000 molecules of alpha polyglutamated methotrexate, or any range therebetween. In some embodiments, the liposome interior space contains between 10,000 to 100,000 molecules of alpha polyglutamated methotrexate, or any range therebetween. In some embodiments, the liposomes are non-pegylated and have an interior space containing less than 500,000 or less than 200,000 molecules of alpha-polyglutamated methotrexate. In some embodiments, the liposomes are non-pegylated and the internal space of the liposomes contains between 10 and 100,000 molecules of alpha polyglutamated methotrexate, or any range therebetween. In other embodiments, the liposome is non-pegylated, and the internal space of the liposome contains between 10,000 and 100,000 molecules of alpha-polyglutamated methotrexate, or any range therebetween. In some embodiments, the liposomes are targeted and non-pegylated (TLp-alpha PMTX) and have an interior space containing less than 500,000 or less than 200,000 alpha polyglutamated methotrexate molecules. In some embodiments, the liposome is targeted and non-pegylated, and the internal space of the liposome contains between 10 and 100,000 molecules of alpha-polyglutamated methotrexate, or any range therebetween. In other embodiments, the liposome is targeted and non-pegylated, and the interior space of the liposome contains between 10,000 and 100,000 molecules of alpha polyglutamated methotrexate, or any range therebetween. In some embodiments, the liposomes are non-targeted and non-pegylated (NTLp- α PMTX) and have an interior space containing less than 500,000 or less than 200,000 α polyglutamated methotrexate molecules. In some embodiments, the liposome is non-targeted and non-pegylated, and the interior space of the liposome contains between 10 and 100,000 molecules of alpha polyglutamated methotrexate, or any range therebetween. In other embodiments, the liposome is non-targeted and non-pegylated, and the interior space of the liposome contains between 10,000 and 100,000 molecules of alpha polyglutamated methotrexate, or any range therebetween.
In some embodiments, the liposome encapsulates alpha polyglutamated methotrexate containing 2-10 glutamyl groups (i.e., Lp-alpha PMTX, including PLp-alpha PMTX, TPLp-alpha PMTX, TLp-alpha PMTX, and NTLp-alpha PMTX), and has an interior space containing less than 500,000 or 200,000 molecules of alpha polyglutamated methotrexate containing 2-10 glutamyl groups. In some embodiments, the liposome interior space contains between 10 and 100,000 molecules of alpha polyglutamated methotrexate containing 2-10 glutamyl groups, or any range therebetween. In other embodiments, the liposome interior space contains between 10,000 and 100,000 molecules of 2-10 glutamyl-containing alpha polyglutamated methotrexate, or any range therebetween. In some embodiments, the liposomes are non-pegylated and have an interior space containing less than 500,000 or 200,000 molecules of alpha polyglutamated methotrexate with 2-10 glutamyl groups. In some embodiments, the liposomes are non-pegylated and the internal space of the liposomes contains between 10 and 100,000 molecules of alpha polyglutamated methotrexate containing 2-10 glutamyl groups, or any range therebetween. In other embodiments, the liposomes are non-pegylated and the internal space of the liposomes contains between 10,000 and 100,000 molecules of alpha polyglutamated methotrexate containing 2-10 glutamyl groups, or any range therebetween. In some embodiments, the liposomes are targeted and non-pegylated (TLp-alpha PMTX) and have an interior space containing less than 500,000 or 200,000 alpha polyglutamated methotrexate molecules with 2-10 glutamyl groups. In some embodiments, the liposomes are targeted and non-pegylated, and the internal space of the liposomes contains between 10 and 100,000 molecules of 2-10 glutamyl-containing alpha polyglutamated methotrexate, or any range therebetween. In other embodiments, the liposome is targeted and non-pegylated, and the interior space of the liposome contains between 10,000 and 100,000 molecules of 2-10 glutamyl-containing alpha polyglutamated methotrexate, or any range therebetween. In some embodiments, the liposomes are non-targeted and non-pegylated (NTLp- α PMTX) and have an interior space containing less than 500,000 or 200,000 molecules of α polyglutamated methotrexate with 2-10 glutamyl groups. In some embodiments, the liposome is non-targeted and non-pegylated, and the interior space of the liposome contains between 10 and 100,000 α polyglutamated methotrexate molecules containing 2-10 glutamyl groups, or any range therebetween. In other embodiments, the liposome is non-targeted and non-pegylated, and the interior space of the liposome contains between 10,000 and 100,000 molecules of 2-10 glutamyl-containing alpha polyglutamated methotrexate, or any range therebetween.
In some embodiments, the liposome encapsulates alpha-tetraglutamated methotrexate (i.e., Lp-alpha PMTX, including PLp-alpha PMTX, TPLp-alpha PMTX, TLp-alpha PMTX, and NTLp-alpha PMTX), and has an interior space containing less than 500,000 or 200,000 molecules of alpha-tetraglutamated methotrexate. In some embodiments, the liposome interior space contains between 10 to 100,000 molecules of α -tetraglutamated methotrexate, or any range therebetween. In some embodiments, the liposome interior space contains between 10,000 to 100,000 molecules of α -tetraglutamated methotrexate, or any range therebetween. In some embodiments, the liposomes are non-pegylated and have an interior space containing less than 500,000 or 200,000 molecules of alpha-tetraglutamated methotrexate. In some embodiments, the liposomes are non-pegylated and the internal space of the liposomes contains between 10 and 100,000 molecules of alpha tetraglutamated methotrexate, or any range therebetween. In other embodiments, the liposome is non-pegylated, and the internal space of the liposome contains between 10,000 and 100,000 molecules of alpha-tetraglutamated methotrexate, or any range therebetween. In some embodiments, the liposomes are targeted and non-pegylated (TLp-alpha PMTX) and have an interior space containing less than 500,000 or 200,000 alpha tetraglutamated methotrexate molecules. In some embodiments, the liposome is targeted and non-pegylated, and the internal space of the liposome contains between 10 and 100,000 molecules of alpha-tetraglutamated methotrexate, or any range therebetween. In other embodiments, the liposome is targeted and non-pegylated, and the internal space of the liposome contains between 10,000 and 100,000 molecules of alpha-tetraglutamated methotrexate, or any range therebetween. In some embodiments, the liposomes are non-targeted and non-pegylated (NTLp- α PMTX) and have an interior space containing less than 500,000 or 200,000 α tetraglutamated methotrexate molecules. In some embodiments, the liposome is non-targeted and non-pegylated, and the interior space of the liposome contains between 10 and 100,000 molecules of alpha-tetraglutamated methotrexate, or any range therebetween. In other embodiments, the liposome is non-targeted and non-pegylated, and the interior space of the liposome contains between 10,000 and 100,000 molecules of alpha-tetraglutamated methotrexate, or any range therebetween.
In some embodiments, the liposome encapsulates alpha pentaglutamated methotrexate (i.e., Lp-alpha PMTX, including PLp-alpha PMTX, TPLp-alpha PMTX, TLp-alpha PMTX, and NTLp-alpha PMTX), and has an interior space containing less than 500,000 or 200,000 molecules of alpha pentaglutamated methotrexate. In some embodiments, the liposome interior space contains between 10 and 100,000 molecules of alpha pentaglutamated methotrexate, or any range therebetween. In some embodiments, the liposome interior space contains between 10,000 to 100,000 molecules of alpha pentaglutamated methotrexate, or any range therebetween. In some embodiments, the liposome is non-pegylated, and has an interior space containing less than 500,000 or 200,000 molecules of alpha pentaglutaminated methotrexate. In some embodiments, the liposome is non-pegylated, and the internal space of the liposome contains between 10 and 100,000 molecules of alpha pentaglutaminated methotrexate, or any range therebetween. In other embodiments, the liposome is non-pegylated, and the internal space of the liposome contains between 10,000 and 100,000 molecules of alpha pentaglutaminated methotrexate, or any range therebetween. In some embodiments, the liposomes are targeted and non-pegylated (TLp-alpha PMTX) and have an interior space containing less than 500,000 or 200,000 alpha pentaglutamated methotrexate molecules. In some embodiments, the liposome is targeted and non-pegylated, and the internal space of the liposome contains between 10 and 100,000 molecules of alpha pentaglutaminated methotrexate, or any range therebetween. In other embodiments, the liposome is targeted and non-pegylated, and the internal space of the liposome contains between 10,000 and 100,000 molecules of alpha pentaglutamated methotrexate, or any range therebetween. In some embodiments, the liposomes are non-targeted and non-pegylated (NTLp- α PMTX) and have an interior space containing less than 500,000 or 200,000 α pentaglutamated methotrexate molecules. In some embodiments, the liposome is non-targeted and non-pegylated, and the interior space of the liposome contains between 10 and 100,000 molecules of alpha pentaglutamated methotrexate, or any range therebetween. In other embodiments, the liposome is non-targeted and non-pegylated, and the interior space of the liposome contains between 10,000 and 100,000 molecules of alpha penta-glutamated methotrexate, or any range therebetween.
In some embodiments, the liposome encapsulates alpha-hexaglutamated methotrexate (i.e., Lp-alpha PMTX, including PLp-alpha PMTX, TPLp-alpha PMTX, TLp-alpha PMTX, and NTLp-alpha PMTX), and has an interior space containing less than 500,000 or 200,000 molecules of alpha-hexaglutamated methotrexate. In some embodiments, the liposome interior space contains between 10 and 100,000 molecules of α -hexaglutaminated methotrexate, or any range therebetween. In other embodiments, the liposome interior space contains between 10,000 and 100,000 molecules of α -hexaglutaminated methotrexate, or any range therebetween. In some embodiments, the liposome is non-pegylated, and has an interior space containing less than 500,000 or 200,000 molecules of α -hexaglutaminated methotrexate. In some embodiments, the liposome is non-pegylated, and the internal space of the liposome contains between 10 and 100,000 molecules of α -hexaglutaminated methotrexate, or any range therebetween. In other embodiments, the liposome is non-pegylated, and the internal space of the liposome contains between 10,000 and 100,000 molecules of α -hexamethy methotrexate, or any range therebetween. In some embodiments, the liposomes are targeted and non-pegylated (TLp-alpha PMTX) and have an interior space containing less than 500,000 or 200,000 alpha hexaglutaminated methotrexate molecules. In some embodiments, the liposome is targeted and non-pegylated, and the internal space of the liposome contains between 10 and 100,000 molecules of α -hexamethy methotrexate, or any range therebetween. In other embodiments, the liposome is targeted and non-pegylated, and the internal space of the liposome contains between 10,000 and 100,000 molecules of α -hexamethinized methotrexate, or any range therebetween. In some embodiments, the liposomes are non-targeted and non-pegylated (NTLp- α PMTX) and have an interior space containing less than 500,000 or 200,000 α hexaglutamated methotrexate molecules. In some embodiments, the liposome is non-targeted and non-pegylated, and the interior space of the liposome contains between 10 and 100,000 molecules of α -hexamethy methotrexate, or any range therebetween. In other embodiments, the liposome is non-targeted and non-pegylated, and the interior space of the liposome contains between 10,000 and 100,000 molecules of α -hexamethinized methotrexate, or any range therebetween.
In some embodiments, the present disclosure provides a liposomal alpha-polyglutamated methotrexate composition, wherein the liposomes encapsulate the alpha-polyglutamated methotrexate or a salt or acid thereof, and one or more pharmaceutically acceptable aqueous carriers. In some embodiments, the liposome interior space contains trehalose. In some embodiments, the liposome interior space contains 1% to 50% trehalose by weight. In some embodiments, the liposome interior space contains HBS at a concentration of between 1 and 200mM and a pH of between 2 and 8. In some embodiments, the liposome interior space has a pH of 5-8, or any range therebetween. In some embodiments, the liposome interior space has a pH of 6-7, or any range therebetween. In some embodiments, the liposome interior space has a total concentration of sodium acetate and calcium acetate between 50mM to 500mM or any range therebetween.
A non-polyglutamated antifolate agent
In some embodiments, the liposomal alpha polyglutamated methotrexate (i.e., Lp-alpha PMTX, including PLp-alpha PMTX, TPLp-alpha PMTX, TLp-alpha PMTX, and NTLp-alpha PMTX) composition comprises alpha polyglutamated methotrexate (e.g., alpha PMTX disclosed herein) and one or more non-polyglutamated polyglutamable antifolate compositions.
In some embodiments, the Lp-a PMTX (e.g., PLp-a PMTX, TPLp-a PMTX, TLp-a PMTX, and NTLp-a PMTX) comprises alpha polyglutamated methotrexate (e.g., a PMTX disclosed herein) and Methotrexate (MTX). In some embodiments, the Lp-a PMTX (i.e., liposomal a polyglutamated methotrexate) comprises a polyglutamated methotrexate and a polyglutamated antifolate selected from the group consisting of: methotrexate (MTX), Pemetrexed (PMX), Lometrexol (LMX), Raltitrexed (RTX), pralatrexate, AG2034, GW1843, aminopterin and LY 309887. In some embodiments, the Lp-a PMTX comprises alpha polyglutamated methotrexate and lometrexol. In some embodiments, the Lp-a PMTX comprises alpha polyglutamated methotrexate and pemetrexed. In some embodiments, the Lp-a PMTX comprises alpha polyglutamated methotrexate and folinic acid. In some embodiments, the Lp- α PMTX comprises α polyglutamated methotrexate and a triazine antifolate derivative (e.g., sulfonyl fluorotriazine, such as NSC 127755). In some embodiments, the Lp- α PMTX comprises α polyglutamated methotrexate and a serine hydroxymethyltransferase (SHMT2) inhibitor. In some embodiments, the SHMT2 inhibitor is an antifolate (e.g., a polyglutamated or non-polyglutamated antifolate). In some embodiments, the SHMT2 inhibitor is an antifolate.
B non-polyglutamated antifolate agent
In some embodiments, the Lp- α PMTX (e.g., PLp- α PMTX, TPLp- α PMTX, TLp- α PMTX, and NTLp- α PMTX) comprises α polyglutamated methotrexate (e.g., α PMTX disclosed herein) and a so-called "non-polyglutamated" antifolate. In some embodiments, the liposomes comprise alpha polyglutamated methotrexate and a non-polyglutamated antifolate that inhibits one or more enzymes of the folate cycle metabolic pathway. In other embodiments, the non-polyglutamated antifolate inhibits one or more enzymes selected from the group consisting of: thymidylate Synthase (TS), dihydrofolate reductase (DHFR), glycinamide ribonucleotide (GAR) converting enzyme, and aminoimidazole carboxamide ribonucleotide (AICAR) converting enzyme. In some embodiments, the liposomes comprise alpha polyglutamated methotrexate and a non-polyglutamated antifolate that inhibits DHFR. In some embodiments, the liposomes comprise alpha polyglutamated methotrexate and a non-polyglutamated antifolate that inhibits TS. In some embodiments, the liposome comprises alpha polyglutamated methotrexate and a non-polyglutamated antifolate that inhibits a GAR or AICAR converting enzyme. In other embodiments, the non-polyglutamated antifolate agent is selected from the group consisting of: trimetrexate (TMQ), pirtrexin (BW301U), and talotrexin (PT 523). In other embodiments, the non-polyglutamated antifolate agent is selected from the group consisting of: nolatrexed (AG337), prallexed (ZD9331, BGC9331), and BGC 945(ONX 0801).
C platinum
In some embodiments, the liposomes comprise alpha polyglutamated methotrexate (Lp-alpha PMTX, e.g., PLp-alpha PMTX, TPLp-alpha PMTX, TLp-alpha PMTX, and NTLp-alpha PMTX), comprise alpha polyglutamated methotrexate (e.g., alpha PMTX disclosed herein), and a platinum-based chemotherapeutic agent or a salt or acid thereof. In some embodiments, the liposome comprises an alpha polyglutamated methotrexate/platinum-based agent complex (e.g., as described in section IIC).
In some embodiments, the Lp-a PMTX comprises a platinum-based chemotherapeutic agent selected from the group consisting of: cisplatin, carboplatin, and oxaliplatin, or salts or acids thereof. In other embodiments, the Lp- α PMTX comprises an analog of a platinum-based chemotherapeutic agent selected from the group consisting of: cisplatin, carboplatin, or oxaliplatin, or a salt or acid thereof.
In some embodiments, the Lp-a PMTX comprises α polyglutamated methotrexate and cisplatin, or a salt or acid thereof. In some embodiments, the Lp- α PMTX comprises α polyglutamated methotrexate and a cisplatin analog or salt or acid thereof.
In some embodiments, the Lp-a PMTX comprises alpha polyglutamated methotrexate and carboplatin, or salts or acids thereof. In some embodiments, the liposomes comprise alpha polyglutamated methotrexate and a carboplatin analog or salt or acid thereof.
In some embodiments, the Lp- α PMTX comprises α polyglutamated methotrexate and oxaliplatin, or a salt or acid thereof. In some embodiments, the liposome comprises alpha polyglutamated methotrexate and an oxaliplatin analog or salt or acid thereof.
In some embodiments, the liposomes comprise alpha polyglutamated methotrexate (e.g., alpha PMTX disclosed herein) and a platinum-based chemotherapeutic agent selected from the group consisting of: nedaplatin, heptaplatin and lobaplatin, or salts or acids thereof. In some embodiments, the Lp-a PMTX comprises an analog of a polyglutamated methotrexate and a platinum-based chemotherapeutic agent selected from the group consisting of: nedaplatin, heptaplatin and lobaplatin, or salts or acids thereof.
In some embodiments, the Lp-a PMTX comprises alpha polyglutamated methotrexate and a platinum-based chemotherapeutic agent selected from the group consisting of: satraplatin, carboplatin, cisplatin, cycloplatin, dexormaplatin, spiroplatin, picoplatin, triplatin, tetraplatin, iproplatin, ormaplatin, zeniplatin, platinum-triamine, traplatin, enloplatin, JM216, 254-S, NK121, CI-973, DWA 2114R, NDDP, and dedaplatin, or salts or acids thereof. In some embodiments, the Lp-a PMTX comprises an analog of a polyglutamated methotrexate and a platinum-based chemotherapeutic agent selected from the group consisting of: satraplatin, carboplatin, cisplatin, cycloplatin, dexormaplatin, spiroplatin, picoplatin, triplatin, tetraplatin, iproplatin, ormaplatin, zeniplatin, platinum-triamine, traplatin, enloplatin, JM216, 254-S, NK121, CI-973, DWA 2114R, NDDP, and dedaplatin, or salts or acids thereof.
In some embodiments, the liposome composition comprises a liposome further comprising one or more of an immunostimulatory agent, a detectable label, and a maleimide disposed on at least one of the PEG and an exterior of the liposome.
D Cyclodextrin
In further embodiments, the α PMTX liposome comprises α PMTX (e.g., α PMTX disclosed herein) and a cyclodextrin (e.g., cyclodextrin in section IIC herein).
In some embodiments, the alpha PMTX liposomes comprise a complex formed from a cyclodextrin and a therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic compound or a salt or acid thereof. In another embodiment, the therapeutic agent is a chemotherapeutic agent or a salt or acid thereof. In another embodiment, the therapeutic agent is a platinum-based drug. In another embodiment, the therapeutic agent is a taxane-based drug. In additional embodiments, the therapeutic agent of the cyclodextrin/therapeutic agent complex is a member selected from the group consisting of: gemcitabine, gemcitabine-based therapeutics, doxorubicin, antifolates, antifolate-based chemotherapeutics or salts or acid, acid or free base forms thereof. In further embodiments, the complex has a cyclodextrin/therapeutic agent molar ratio in the range of 1-10: 1. In some embodiments, the molar ratio of alpha PMTX/therapeutic agent in the complex is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1 (21-50), or 1: > 50. In some embodiments, the complex has a cyclodextrin/therapeutic agent molar ratio of: 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1 or >50: 1.
In some embodiments, the alpha PMTX liposome comprises alpha PMTX and a cyclodextrin/platinum-based chemotherapeutic agent complex. In some embodiments, the platinum-based chemotherapeutic agent is selected from the group consisting of: cisplatin, carboplatin, and oxaliplatin, or salts or acids thereof. In other embodiments, the cyclodextrin/platinum-based chemotherapeutic agent complex comprises an analog of cisplatin, carboplatin, oxaliplatin, or a salt or acid thereof. In some embodiments, the complex has a cyclodextrin/platinum-based agent molar ratio in the range of 1-10: 1. In some embodiments, the complex has a cyclodextrin/platinum-based agent molar ratio of 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1 (21-50), or 1: > 50. In some embodiments, the mole ratio of alpha PMTX/platinum-based agent in the composite is: 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1 or >50: 1.
In some embodiments, the platinum-based chemotherapeutic agent is selected from the group consisting of: cisplatin, carboplatin, and oxaliplatin, or salts or acids thereof. In other embodiments, the cyclodextrin/platinum-based chemotherapeutic agent complex comprises an analog of cisplatin, carboplatin, oxaliplatin, or a salt or acid thereof. In some embodiments, the complex has a cyclodextrin/platinum-based agent molar ratio in the range of 1-10: 1. In some embodiments, the complex has a cyclodextrin/platinum-based agent molar ratio of 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1 (21-50), or 1: > 50. In some embodiments, the molar ratio of alpha PMTX/platinum-based chemotherapeutic agent in the complex is: 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1 or >50: 1.
In further embodiments, the cyclodextrin// platinum-based agent complex is encapsulated in a liposome (e.g., as described herein or otherwise known in the art).
In other embodiments, the present disclosure provides a complex comprising cyclodextrin and cisplatin or a salt or acid thereof. In some embodiments, the complex has a cyclodextrin/cisplatin (or cisplatin salt or acid) molar ratio in the range of 1-10: 1. In some embodiments, the cyclodextrin/cisplatin (or cisplatin salt or acid) molar ratio in the complex is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1 (21-50), or 1: > 50. In some embodiments, the molar ratio of alpha PMTX/cisplatin (or cisplatin salt or acid) in the complex is: 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1 or >50: 1. In further embodiments, the cyclodextrin// cisplatin (or cisplatin salt or acid) complex is encapsulated in a liposome (e.g., as described herein or otherwise known in the art).
In another embodiment, the present disclosure provides a complex comprising a cyclodextrin and carboplatin or a salt or acid thereof. In some embodiments, the complex has a cyclodextrin/carboplatin (or carboplatin salt or acid) molar ratio in the range of 1-10: 1. In some embodiments, the molar ratio of cyclodextrin/carboplatin (or carboplatin salt or acid) in the complex is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1 (21-50), or 1: > 50. In some embodiments, the molar ratio of alpha PMTX/carboplatin (or carboplatin salt or acid) in the composite is: 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1 or >50: 1. In further embodiments, the cyclodextrin/carboplatin (or carboplatin salt or acid) complex is encapsulated in a liposome (e.g., as described herein or otherwise known in the art).
In another embodiment, the present disclosure provides a complex comprising cyclodextrin and oxaliplatin or a salt or acid thereof. In some embodiments, the cyclodextrin/oxaliplatin (or oxaliplatin salt or acid) molar ratio in the complex is in the range of 1-10: 1. In some embodiments, the cyclodextrin/oxaliplatin (or oxaliplatin salt or acid) molar ratio in the complex is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1 (21-50), or 1: > 50. In some embodiments, the molar ratio of alpha PMTX/oxaliplatin (or oxaliplatin salt or acid) in the complex is: 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1 or >50: 1. In further embodiments, the cyclodextrin/oxaliplatin (or oxaliplatin salt or acid) complex is encapsulated in a liposome (e.g., as described herein or otherwise known in the art).
In additional embodiments, the present disclosure provides a complex comprising cyclodextrin and a platinum-based chemotherapeutic selected from the group consisting of nedaplatin, heptaplatin, lobaplatin, satraplatin, carboplatin, cisplatin, cycloplatin, dexormaplatin, spiroplatin, picoplatin, triplatin, tetraplatin, iproplatin, ormaplatin, zeniplatin, platinum-triamine, traplatin, enloplatin, JM216, NK121, CI973, DWA 211R, NDDP, and dedaplatin, or salts or acids thereof. The complex has a cyclodextrin/oxaliplatin (or oxaliplatin salt or acid) molar ratio in the range of 1-10: 1. In some embodiments, the complex has a cyclodextrin/platinum-based chemotherapeutic (or a salt or acid or analog thereof) molar ratio of 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1 (21-50), or 1: > 50. In some embodiments, the molar ratio of alpha PMTX/platinum-based chemotherapeutic agent (or a salt or acid or analog thereof) in the complex is: 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1 or >50: 1. In additional embodiments, the cyclodextrin/platinum-based chemotherapeutic agent (or salt or acid or analog thereof) complex is encapsulated in a liposome (e.g., as described herein or otherwise known in the art).
In some embodiments, the present disclosure provides a composition comprising a cyclodextrin/taxane-based chemotherapeutic agent complex. In some embodiments, the taxane-based chemotherapeutic agent is selected from the group consisting of: paclitaxel (PTX), Docetaxel (DTX), Larotaxel (LTX) and Cabazitaxel (CTX), or salts or acids thereof. In some embodiments, the complex has a cyclodextrin/taxane-based agent molar ratio in the range of 1-10: 1. In some embodiments, the complex has a cyclodextrin/taxane-based agent molar ratio of 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1 (21-50), or 1: > 50. In some embodiments, the molar ratio of alpha PMTX/taxane-based agent in the complex is: 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1 or >50: 1. In further embodiments, the cyclodextrin// taxane-based agent complex is encapsulated in a liposome (e.g., as described herein or otherwise known in the art).
In additional embodiments, the present disclosure provides a complex comprising a cyclodextrin and Paclitaxel (PTX) or a salt or acid thereof. In other embodiments, the cyclodextrin/taxane-based chemotherapeutic complex comprises an analog of Paclitaxel (PTX) or a salt or acid thereof. In some embodiments, the complex has a cyclodextrin/paclitaxel (or paclitaxel salt or acid) molar ratio in the range of 1-10: 1. In some embodiments, the cyclodextrin/paclitaxel (or paclitaxel salt or acid) molar ratio in the complex is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1 (21-50), or 1: > 50. In some embodiments, the molar ratio of α PMTX/paclitaxel (or paclitaxel salt or acid) in the complex is: 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1 or >50: 1. In additional embodiments, the cyclodextrin/paclitaxel (or paclitaxel salt or acid) complex is encapsulated in a liposome (e.g., as described herein or otherwise known in the art).
In additional embodiments, the present disclosure provides a complex comprising cyclodextrin and Docetaxel (DTX) or a salt or acid thereof. In other embodiments, the cyclodextrin/taxane-based chemotherapeutic complex comprises an analog of Docetaxel (DTX) or a salt or acid thereof. In some embodiments, the complex has a cyclodextrin/docetaxel (or docetaxel salt or acid) molar ratio in the range of 1-10: 1. In some embodiments, the complex has a cyclodextrin/docetaxel (or docetaxel salt or acid) molar ratio of 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1 (21-50), or 1: > 50. In some embodiments, the molar ratio of alpha PMTX/docetaxel (or docetaxel salt or acid) in the complex is: 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1 or >50: 1. In additional embodiments, the cyclodextrin/docetaxel (or docetaxel salt or acid) complex is encapsulated in a liposome (e.g., as described herein or otherwise known in the art).
In additional embodiments, the present disclosure provides a complex comprising a cyclodextrin and ralfataxel (LTX) or a salt or acid thereof. In other embodiments, the cyclodextrin/taxane-based chemotherapeutic complex comprises an analog of ralfataxel (LTX) or a salt or acid thereof. In some embodiments, the complex has a cyclodextrin/raloxitol (or raloxitol salt or acid) molar ratio in the range of 1-10: 1. In some embodiments, the molar ratio of cyclodextrin/raloxitol (or raloxitol salt or acid) in the complex is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1 (21-50), or 1: > 50. In some embodiments, the molar ratio of alpha PMTX/raloxitol (or raloxitol salt or acid) in the composite is: 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1 or >50: 1. In additional embodiments, the cyclodextrin/raloxitol (or raloxitol salt or acid) complex is encapsulated in a liposome (e.g., as described herein or otherwise known in the art).
In additional embodiments, the present disclosure provides a complex comprising cyclodextrin and Cabazitaxel (CTX) or salts or acids thereof. In other embodiments, the cyclodextrin/taxane-based chemotherapeutic complex comprises an analog of Cabazitaxel (CTX) or a salt or acid thereof. In some embodiments, the complex has a cyclodextrin/cabazitaxel (or cabazitaxel salt or acid) molar ratio in the range of 1-10: 1. In some embodiments, the complex has a molar ratio of cyclodextrin/cabazitaxel (or cabazitaxel salt or acid) of 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1 (21-50), or 1: > 50. In some embodiments, the molar ratio of alpha PMTX/cabazitaxel (or cabazitaxel salt or acid) in the complex is: 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1 or >50: 1. In additional embodiments, the cyclodextrin/cabazitaxel (or cabazitaxel salt or acid) complexes are encapsulated in liposomes (e.g., as described herein or otherwise known in the art).
The cyclodextrin of the cyclodextrin/therapeutic agent complex may be derivatized or underivatized. In some embodiments, the cyclodextrin is derivatized. In other embodiments, the cyclodextrin is a derivatized β -cyclodextrin (e.g., hydroxypropyl β -cyclodextrin (HP- β -CD) and sulfobutyl ether β -CD (SBE) - β -cyclodextrin). In some embodiments, the cyclodextrin of the cyclodextrin/therapeutic agent complex is a derivatized β -cyclodextrin comprising: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more 2-hydroxypropyl-3-group substitutions of hydroxyl; or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more sulfoalkyl ether groups of a hydroxyl group. In other embodiments, the cyclodextrin of the cyclodextrin/therapeutic agent complex is a derivatized β -cyclodextrin comprising: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more sulfobutyl ether groups of hydroxyl groups.
In some embodiments, the cyclodextrin of the cyclodextrin/therapeutic agent complex contained in the alpha PMTX liposome composition is a derivatized cyclodextrin of formula I:
Figure BDA0002715283060000641
wherein:n is 4, 5 or 6; and wherein R1、R2、R3、R4、R5、R7、R8And R9Each independently is-H, straight or branched chain C 1-C8-alkylene, 2-hydroxypropyl-3-group; or optionally substituted straight or branched C1-C6Group, wherein R1、R2、R3、R4、R5、R6、R7、R8And R9At least one of which is a straight or branched chain C1-C8An alkylene or 2-hydroxypropyl-3-group.
In some embodiments, the cyclodextrin of the cyclodextrin/therapeutic agent complex contained in the alpha PMTX liposome composition is a derivatized cyclodextrin of formula II:
Figure BDA0002715283060000642
wherein: n is 4, 5 or 6; and wherein R1、R2、R3、R4、R5、R6、R7、R8And R9Each independently is-O-or-O- (C)2-C6Alkylene) -SO3-A group; wherein R is1And R2At least one of which is independently-O- (C)2-C6Alkylene) -SO3 -A group; and S1、S2、S3、S4、S5、S6、S7、S8And S9Each independently is-H or a pharmaceutically acceptable cation. In other embodiments, wherein the pharmaceutically acceptable cation is selected from the group consisting of: alkali metals, e.g. Li+、Na+Or K+(ii) a Alkaline earth metals, e.g. Ca+2Or Mg+2And ammonium ions and amine cations, such as the cations of (C1-C6) -alkylamines, piperidines, pyrazines, (C1-C6) -alkanolamines and (C4-C8) -cycloalkanolamines.
In some embodiments, the alpha PMTX liposomes comprise between 100 to 100,000 cyclodextrin/therapeutic agent complexes.
In some embodiments, the cyclodextrin derivative of the alpha PMTX/cyclodextrin complex and/or the cyclodextrin/therapeutic agent complex is any one of U.S. patent nos. 6,133,248, 5,874,418, 6,046,177, 5,376,645, 5,134,127, 7,034,013, 6,869,939; and the cyclodextrins disclosed in International application publication No. WO02005/117911, the contents of each of which are incorporated herein by reference in their entirety for all purposes.
In some embodiments, the cyclodextrin derivative of the cyclodextrin/therapeutic agent complex is a sulfoalkyl ether cyclodextrin. In some embodiments, the cyclodextrin derivative of the complex is sulfobutyl ether-3-cyclodextrin, e.g.
Figure BDA0002715283060000644
(CyDex Pharma.Inc., Lenexa, Kansas. methods for preparing sulfobutyl ether-3-cyclodextrins and other sulfoalkyl ether cyclodextrins are known in the art.
In some embodiments, the cyclodextrin derivative of the cyclodextrin/therapeutic agent complex is a compound of formula III:
Figure BDA0002715283060000643
wherein R is equal to:
(e)(H)21-Xor (- (CH)2)4-SO3Na) x, and x ═ 1.0 to 10.0, 1.0 to 5.0, 6.0 to 7.0, or 8.0 to 10.0;
(f)(H)21-Xor (- (CH)2CH(OH)CH3) x, and x ═ 1.0 to 10.0, 1.0 to 5.0, 6.0 to 7.0, or 8.0 to 10.0;
(g)(H)21-Xor (sulfoalkyl ether) x, and x ═ 1.0 to 10.0, 1.0 to 5.0, 6.0 to 7.0, or 8.0 to 10.0; or
(h)(H)21-XOr (- (CH)2)4-SO3Na) x, and x ═ 1.0-10.0, 1.0-5.0, 6.0-7.0, or 8.0-10.0.
Additional cyclodextrins and cyclodextrin/platinum-based therapeutic agent complexes that can be included in the alpha PMTX liposomes and used in accordance with the disclosed methods are disclosed in U.S. application No. 62/583,432, the contents of which are incorporated herein by reference in their entirety.
In some embodiments, the alpha PMTX liposomes comprise a complex of a cyclodextrin and a platinum-based chemotherapeutic agent or a salt thereof. In some embodiments, the platinum-based chemotherapeutic agent is cisplatin or a cisplatin analog. In some embodiments, the platinum-based chemotherapeutic agent is carboplatin. In additional embodiments, the liposome composition comprises a platinum-based chemotherapeutic agent that is a member selected from the group consisting of: carboplatin, cisplatin, oxaliplatin, satraplatin, picoplatin, nedaplatin, triplatin, tetraplatin, lipoplatin (lipoplatin), lobaplatin, ormaplatin, zeniplatin, platinum-triamine, traplatin, enloplatin, JM-216, 254-S, NK 121, CI-973, DWA 2114R, NDDP, and dedaplatin. In some embodiments, the alpha PMTX liposomes comprise between 100 to 100,000 platinum-based chemotherapeutic agent/CD complexes. In further embodiments, the liposome composition comprises liposomes having a diameter in the range of 20nm to 500nm or 20nm to 200nm or any range therebetween. In some embodiments, the liposomes in the composition comprise between 100 and 100,000 platinum.
(3) Targeted liposomes
In some embodiments, the present disclosure provides a liposomal alpha-polyglutamated methotrexate composition, wherein the liposome comprises alpha-polyglutamated methotrexate and a targeting moiety attached to one or both of PEG and the exterior of the liposome, and wherein the targeting moiety has specific affinity for a surface antigen on a target cell of interest. Such liposomes may be generally referred to herein as "targeted liposomes," e.g., liposomes that include one or more targeting moieties or biodistribution modulators on the surface of the liposome or otherwise attached to the liposome. The targeting moiety of the targeted liposome can be any moiety or agent capable of specifically binding to the desired target (e.g., an antigen target expressed on the surface of a target cell of interest). In one embodiment, the targeted liposome specifically and preferentially binds to a target on the surface of a target cell of interest that internalizes the targeted liposome, and the liposome-encapsulated alpha polyglutamated methotrexate (e.g., alpha pentaglutamated MTX or alpha hexaglutamated MTX) exerts its cytotoxic effect in the target cell of interest. In other embodiments, the target cell is a cancer cell, a tumor cell, or a metastatic cell. In some embodiments, the targeted liposome is pegylated.
The term "attachment" or "linked" refers, for example, to any type of linkage, such as covalent linkage, ionic linkage through hydrophobic interactions (e.g., avidin-biotin), and linkage through a functional group such as maleimide or a linker such as PEG. For example, detectable labels, steric stabilizers, liposomes, liposome components, immunostimulants can be directly linked to each other through maleimide functional groups or PEG-maleimide groups.
The composition and source of the targeting moiety is not limiting to the scope of the present disclosure. In some embodiments, the targeting moiety attached to the liposome is a polypeptide or peptidomimetic ligand. Peptide and peptidomimetic targeting moieties include those having naturally occurring or modified peptides, such as D or L peptides; an alpha, beta or gamma peptide; an N-methyl peptide; an azapeptide; peptides having one or more amides, i.e., peptides in which the linkage is replaced with one or more urea, thiourea, carbamate, or sulfonylurea linkages; or a cyclic peptide. Peptidomimetics are molecules that can fold into a defined three-dimensional structure similar to a natural peptide. In some embodiments, the peptide or peptidomimetic targeting moiety is 2-50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.
In some embodiments, the targeting moiety polypeptide is at least 40 amino acid residues in length. In other embodiments, the targeting moiety polypeptide is at least 50, 60, 75, 100, 125, 150, 175, 200, 250, or 300 amino acid residues in length.
In further embodiments, the targeting moiety polypeptide, such as an antibody or antigen binding antibody fragment, is at 0.5x 10-10To 10x 10-6Equilibrium dissociation constant (Kd) in a range to bind to a target antigen, e.g., using
Figure BDA0002715283060000651
The assay is analyzed.
In some embodiments, the targeting moiety is an antibody or antibody derivative. In other embodiments, the binding domain of the targeting moiety polypeptide is not derived from the antigen binding domain of an antibody. In some embodiments, the targeting moiety is a polypeptide derived from a binding scaffold selected from the group consisting of: DARPin, affilin and armadillo repeats, a D domain (see, e.g., WO 2016/164308), a Z domain (Affibody), an adnectin, lipocalin, affilin, anticalin, knottin, fynomer, atrimer, kunitz domain (see, e.g., WO 2004/063337), CTLA4, or avimer (see, e.g., U.S. publication nos. 2004/0175756, 2005/0053973, 2005/0048512, and 2006/0008844).
In further embodiments, the targeting moiety is an antibody or a derivative of the antigen binding domain of an antibody having specific affinity for an epitope on a target cell surface antigen expressed on the surface of a target cell. In some embodiments, the targeting moiety is a full length antibody. In some embodiments, the targeting moiety is an antigen binding portion of an antibody. In some embodiments, the targeting moiety is a scFv. In other embodiments, the targeting moiety is a Fab. In some embodiments, the targeting moiety comprises a binding domain derived from an antigen binding domain of an antibody (e.g., scFv, Fab ', F (ab')2, Fv fragments, disulfide linked Fv (sdFv), Fd fragments consisting of VH and CH1 domains, scFv, minibody, BiTE, Tandab, diabody ((VL-VH)2Or (VH-VL)2) Single domain antibodies (e.g., sdabs, such as nanobodies (VL or VH)) and camelid VHH domains). In some embodiments, the targeting moiety comprises one or more Complementarity Determining Regions (CDRs) derived from an antibody. Examples of suitable antibody-based targeting moieties for the disclosed targeted liposomes include fully human antibodies, humanized antibodies, chimeric antibodies, antibodies The antigen-binding fragment of (a), a single chain antibody, a single domain antibody, a bispecific antibody, a synthetic antibody, a pegylated antibody, and a multimeric antibody. The provided liposome-targeting antibodies can have a combination of the above features. For example, humanized antibodies may be antigen-binding fragments, and may also be pegylated and multimerized.
The term "humanized antibody" refers to a form of a non-human (e.g., murine) antibody that is a specific immunoglobulin chain, chimeric immunoglobulin, or fragment thereof that contains minimal non-human (e.g., murine) sequences. Typically, humanized antibodies are human immunoglobulins in which residues from the Complementarity Determining Regions (CDRs) are replaced by residues from CDRs of non-human species (e.g., mouse, rat, rabbit, and hamster) having the desired specificity, affinity, and capacity (Jones et al, Nature 321:522-525 (1986); Riechmann et al, Nature 332:323-327 (1988); Verhoeyen et al, Science239:1534-1536 (1988)). In some cases, Fv Framework Region (FR) residues of the human immunoglobulin are replaced by corresponding residues in antibodies from non-human species having the desired specificity, affinity, and capacity. Humanized antibodies can be further modified by substitution of additional residues in the Fv framework regions and/or within the substituted non-human residues to improve and optimize antibody specificity, affinity, and/or capacity. In general, a humanized antibody will comprise substantially all of at least one and typically two or three variable domains comprising all or substantially all of the CDR regions corresponding to a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody may further comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically at least a portion of a human immunoglobulin. Examples of methods for generating humanized antibodies are described in U.S. Pat. nos. 5,225,539 and 5,639,641.
In other embodiments, the targeting moiety has specific affinity for an epitope on a surface antigen of the target cell of interest. In some embodiments, the target cell is a cancer cell. In some embodiments, the target cell is a tumor cell. In other embodiments, the target cell is an immune cell.
In some embodiments, the targeting moiety has specific affinity for an epitope expressed on a tumor cell surface antigen. The term "tumor cell surface antigen" refers to an antigen common to particular hyperproliferative disorders, such as cancer. In some embodiments, the targeting moiety has specific affinity for an epitope of a tumor cell surface antigen that is a Tumor Associated Antigen (TAA). TAAs are antigens found on both tumors and some normal cells. When the immune system is immature and unable to respond, TAAs may be expressed on normal cells during fetal development, or may be present at very low levels on normal cells in general, and at much higher levels on tumor cells. Due to the dynamic nature of tumors, in some cases, tumor cells may express distinct antigens at certain stages, and also express antigens that are also expressed on non-tumor cells at other stages. Thus, the inclusion of a marker as a TAA does not exclude that it is considered a tumor specific antigen. In some embodiments, the targeting moiety has specific affinity for an epitope of a tumor cell surface antigen that is a Tumor Specific Antigen (TSA). TSA is an antigen specific to tumor cells and is not present on other cells in the body. In some embodiments, the targeting moiety has specific affinity for an epitope of a tumor cell surface antigen expressed on the surface of a cancer, including, but not limited to, primary or metastatic melanoma, thymoma, lymphoma, sarcoma, lung cancer (e.g., NSCLC or SCLC), liver cancer, non-hodgkin's lymphoma, leukemia, multiple myeloma, glioblastoma, neuroblastoma, uterine cancer, cervical cancer, renal cancer, thyroid cancer, bladder cancer, renal cancer, mesothelioma, and adenocarcinomas such as breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, colon cancer, and others known in the art. In some embodiments, the targeting moiety has specific affinity for an epitope of a cell surface antigen expressed on the cell surface in the tumor microenvironment (e.g., and antigens such as VEGFR and TIE1 or TIE2 expressed on endothelial cells and macrophages, respectively, or antigens expressed on tumor stromal cells such as cancer-associated fibroblasts (CAF), tumor infiltrating T cells and other leukocytes, and myeloid cells including mast cells, eosinophils, and tumor-associated macrophages (TAMs)).
In some embodiments, a targeted liposomal alpha PMTX composition (e.g., TLp-alpha PMTX or TPLp-alpha PMTX) comprises a targeting moiety that has specific affinity for an epitope of a cancer or tumor cell surface antigen that is preferentially/differentially expressed on a target cell (e.g., a cancer cell or tumor cell) as compared to a normal or non-tumor cell, the epitope being present in the tumor cell but absent or inaccessible on the non-tumor cell. For example, in some cases, tumor antigens are located on the surface of normal and malignant cancer cells, but tumor epitopes are only exposed in cancer cells. As another example, a tumor cell surface antigen may undergo a confirmed change in cancerous state that results in the presence of a cancer cell specific epitope. Targeting moieties having specific affinity for an epitope on a tumor cell surface antigen described herein or otherwise known in the art are useful and are encompassed by the disclosed compositions and methods. In some embodiments, the tumor cell having a tumor cell surface antigen is a cancer cell. Examples of such tumor cell surface antigens include, but are not limited to, folate receptor alpha, folate receptor beta, and folate receptor.
In other embodiments, the targeting moiety comprises a polypeptide targeting moiety, such as an antibody or antigen binding antibody fragment, and the targeting moiety has binding specificity for the folate receptor. In some embodiments, e.g., using
Figure BDA0002715283060000671
Assay determined targeting moiety to be at 0.5x 10-10To 10x 10-6Equilibrium dissociation constants (Kd) within the range bind to folate receptors. In some embodiments, the folate receptor bound by the targeting moiety is one or more folate receptors selected from the group consisting of: folate receptor alpha (FR-. alpha.), folate receptor beta (FR-. beta.), and folate receptor (FR-). In another embodiment, the targeting moiety has specific affinity for at least two antigens selected from the group consisting of folate receptor alpha, folate receptor beta and folate receptor. In another embodimentTargeting moieties to folate receptor alpha; folate receptor beta; and folate receptors have specific affinities.
In some embodiments, the targeting moiety has specific affinity for an epitope of a cell surface antigen that, when bound, internalizes the targeting moiety. Many cell surface antigens that internalize a binding partner, such as an antibody, upon binding are known in the art and are considered to be binding targets for the targeting moieties disclosed herein that are expressed on targeted liposomal alpha PMTX compositions (e.g., TLp-alpha PMTX or TPLp-alpha PMTX).
In some embodiments, the targeting moiety has specific affinity for an epitope of a cell surface antigen selected from the group consisting of: GONMB, TACTD 2(TROP2), CEACAM5, EPCAM, folate receptor (e.g., folate receptor-alpha, folate receptor-beta, or folate receptor-), mucin 1(MUC-1), MUC-6, STEAP1, mesothelin, bindin 4, ENPP3, Guanylate Cyclase C (GCC), SLC44A4, NaPi2B, CD70(TNFSF7), CA9 (carbonic anhydrase), 5T4(TPBG), SLTRK6, SC-16, tissue factor, LIV-1(ZIP6), CGEN-15027, Pcadherin, fibronectin ectodomain B (ED-B), VEGFR2(CD309), tenascin, collagen IV, periostin, endothelin receptor, HER2, HER 72, EGFR, EGFRvIII, FGFR 72, FGFR3, CD ZD3, CD 36ZD 3, CD3, FGFR3, CD 36ZD 3, CD 36ZD 3, CD, CD19, CD20, CD22, CD26, CD27L, CD28, CD30, CD33, CD34, CD37, CD38, CD40, CD44, CD56, CD70, CD74, CD79, CD79b, CD98, CD105, CD133, CD138, cripto, IGF-1R, IGF-2R, IGF, EphA receptor, EphB receptor, EphA R, IGF, EphB R, IGF, EphA R, IGF, EphB R, IGF, EphUFB R, IGF, EphB R, IGF, integrin (for example, integrin. alpha. v. beta.3,. beta.5 or. alpha. v. beta.6), C antigen, Trk, VEGFR 72, PGR, VEGF-PGR, VEGF-R, IGF, PGRE, PGR, VEGF-R, IGF, PGR, and PG.
In some embodiments, the targeting moiety has specific affinity for one or more cell surface antigens (such as neoantigens) derived from or determined to be expressed on a cancer (e.g., tumor) in a particular subject.
In some embodiments, the targeting moiety has specific affinity for an epitope of a cell surface antigen selected from the group consisting of mannose-6-phosphate receptor, transferrin receptor, and Cell Adhesion Molecule (CAM). In other embodiments, the targeting moiety has specific affinity for an epitope of a CAM selected from the group consisting of: intercellular adhesion molecule (ICAM), platelet-endothelial adhesion molecule (PECAM), activated leukocyte adhesion molecule (ALCAM), B-lymphocyte cell adhesion molecule (BL-CAM), Vascular Cell Adhesion Molecule (VCAM), mucosal vascular addressen cell adhesion molecule (MAdCAM), CD44, LFA-2, LFA-3, and basal immunoglobulin.
The Folate Receptor (FR) discussed herein is distinct from the Reducing Folate Carrier (RFC) and utilizes a different pathway to bring folate and antifolates into the cell. In some embodiments, the targeting moiety specifically binds to a folate receptor. In other embodiments, the targeting moiety specifically binds to a folate receptor selected from the group consisting of folate receptor alpha, folate receptor beta, and folate receptor. Antibodies to folate receptor alpha can be routinely generated using techniques known in the art. In addition, the sequences of many anti-folate receptor antibodies are of the public domain and/or are commercially available and readily available.
Murine antibodies directed against the folate receptor are examples of antibodies that can be used as targeting moieties for the disclosed targeted liposomes, are murine antibodies directed against the folate receptor. The sequences of these antibodies are known and described, for example, in U.S. patent nos. 5,646,253; 8,388,972, respectively; 8,871,206, respectively; and 9,133,275, and international application numbers PCT/US2011/056966 and PCT/US 2012/046672. For example, based on sequences already disclosed in the public domain, genes for antibodies can be synthesized and placed in transient expression vectors and antibodies produced in the HEK-293 transient expression system. The antibody can be a whole antibody, a Fab, or any of the various antibody variants discussed herein or known in the art.
In some embodiments, the targeting liposome (e.g., TL- α PMTX or TPL- α PMTX) contains 1 to 1,000 or more than 1,000 targeting moieties on its surface. In some embodiments, the targeted liposomes contain 30 to 1,000, 30 to 500, 30 to 250, or 30-200 targeting moieties or any range therebetween. In some embodiments, the targeted liposome contains less than 220 targeting moieties, less than 200 targeting moieties, or less than 175 targeting moieties. In some embodiments, the targeting moiety is non-covalently bonded to the exterior of the liposome (e.g., via ionic interaction or GPI anchor).
In some embodiments, molecules on the exterior of the targeted liposome (e.g., TL- α PMTX or TPL- α PMTX) include lipids, targeting moieties, steric stabilizers (e.g., PEG), maleimides, and cholesterol. In some embodiments, the targeting moiety is covalently bound via a maleimide functional group. In some embodiments, the targeting moiety is covalently attached to a liposome component or a steric stabilizer, such as a PEG molecule. In some embodiments, all targeting moieties of the liposome are bound to one component of the liposome, such as PEG. In other embodiments, the targeting moiety of the targeted liposome is bound to a different component of the liposome. For example, some targeting moieties may be conjugated to a lipid component or cholesterol, some targeting moieties may be conjugated to a steric stabilizer (e.g., PEG), and other targeting moieties may be conjugated to a detectable label or another targeting moiety. In some embodiments, the exterior of the targeted liposome (e.g., TL-alpha PMTX or TPL-alpha PMTX) further comprises one or more of an immunostimulatory agent, a detectable label, and a maleimide disposed on at least one of the PEG and the exterior of the liposome.
In some embodiments, the targeted liposome (e.g., TL- α PMTX or TPL- α PMTX) is anionic or neutral. In some embodiments, the targeted anionic or neutral liposomes have a diameter in the range of 20nm to 500nm or 20nm to 200nm or any range therebetween. In other embodiments, the targeted anionic or neutral liposomes have a diameter in the range of 80nm to 120nm or any range therebetween.
In other embodiments, the targeting liposome (e.g., TL-alpha PMTX or TPL-alpha PMTX) is cationic. In some embodiments, the targeted anionic or neutral liposomes have a diameter in the range of 20nm to 500nm or 20nm to 200nm or any range therebetween. In other embodiments, the targeted anionic or neutral liposomes have a diameter in the range of 80nm to 120nm or any range therebetween.
In some embodiments, the targeted liposome comprises at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75% (w/w) alpha polyglutamated MTX. In some embodiments, at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75% of the starting material of alpha polyglutamated MTX is encapsulated (embedded) in the targeted liposomes during the process of making the targeted liposomes.
In some embodiments, the targeted liposome composition comprises 30% -70%, 30% -60%, or 30% -50% (w/w) of α -tetraglutamated MTX, or any range therebetween. In some embodiments, the targeted liposome comprises at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75% (w/w) alpha tetraglutamated MTX. In some embodiments, at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75% of the starting material of alpha tetraglutamylated MTX is encapsulated (embedded) in the targeted liposome during the process of preparing the targeted liposome.
In some embodiments, the targeted liposome composition comprises 30% -70%, 30% -60%, or 30% -50% (w/w) of α pentaglutamated MTX, or any range therebetween. In some embodiments, the targeted liposome comprises at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75% (w/w) of α pentaglutamated MTX. In some embodiments, at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75% of the starting material of alpha pentaglutamated MTX is encapsulated (embedded) in the targeted liposome during the process of making the targeted liposome.
In some embodiments, the targeted liposome composition comprises 30% -70%, 30% -60%, or 30% -50% (w/w) of α -hexaglutamated MTX, or any range therebetween. In some embodiments, the targeted liposome comprises at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75% (w/w) of α -hexaglutamated MTX. In some embodiments, at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75% of the starting material of alpha hexaglutamated MTX is encapsulated (embedded) in the targeted liposome during the process of making the targeted liposome.
Methods and techniques for covalently associating a polypeptide targeting moiety with a liposome surface molecule are known in the art and can be readily used to prepare TL- α PMTX or TPL- α PMTX liposome compositions.
Chemical binding of non-protein targeting moieties and other compositions to the surface of liposomes can be employed. Thus, the non-protein moiety may be covalently or non-covalently attached to, entrapped in, or adsorbed onto the liposome using any attachment or conjugation method known in the art and/or any suitable chemical linker. The exact type and chemical nature of such cross-linking agents and cross-linking methods are preferably adapted to the type of affinity groups used and the nature of the liposomes. Methods for binding or adsorbing or linking targeting moieties are also well known in the art. For example, in some embodiments, the targeting moiety may be attached to the group at the interface via, but not limited to, a polar group such as amino, SH, hydroxyl, aldehyde, formyl, carboxyl, His-tag, or other polypeptide. In addition, the targeting moiety can be attached via, but is not limited to, an active group such as succinimidyl succinate, cyanuric chloride, tosyl activating group, imidazole group, CNBr, NHS, activated CH, ECH, EAH, epoxy, thiopropyl, activated thiol, and the like. Furthermore, the targeting moiety may be attached via, but is not limited to, hydrophobic bonds (van der waals forces) or electrostatic interactions (e.g., dianions, polyanions, poly-cations, etc.) that may or may not include a cross-linking agent.
(4) Production of liposomes
In some embodiments, the present disclosure provides a method of making a liposome composition disclosed herein. In one embodiment, the method comprises forming a mixture comprising: (1) a liposome component; and (2) alpha polyglutamated (e.g., penta-or hexa-glutamated) methotrexate in aqueous solution. In other embodiments, the mixture comprises a pegylated liposome component. The mixture is then homogenized to form liposomes in aqueous solution. In addition, the mixture can be extruded through a membrane to form liposomes that encapsulate the α -polyglutamated methotrexate in an aqueous solution. It is to be understood that the liposomal composition of the present disclosure may comprise any lipid (including cholesterol), including functionalized lipids and lipids linked to a targeting moiety, a detectable label, and a steric stabilizer, or any subset of all of these. It should also be noted that the biologically active alpha polyglutamated methotrexate in aqueous solution may comprise any of the reagents and chemicals discussed herein or otherwise known in the art either internal or external to the liposomes, including, for example, buffers, salts, and cryoprotectants.
In some embodiments, the present disclosure provides a method of making a targeted pegylated liposomal alpha polyglutamated methotrexate (targeted PLp-alpha PMTX) or non-targeted PLp-alpha PMTX disclosed herein. In one embodiment, the method comprises forming a mixture comprising: (1) a liposome component; (2) α polyglutamated (e.g., penta-or hexa-glutamated) methotrexate in aqueous solution; and (3) a targeting moiety. The mixture is then homogenized to form liposomes in aqueous solution. In addition, the mixture can be extruded through a membrane to form liposomes that encapsulate the targeted alpha-polyglutamated methotrexate in an aqueous solution. It is to be understood that the targeted pegylated liposome component may comprise any lipid (including cholesterol), including functionalized lipids and lipids linked to a targeting moiety, a detectable label, and a steric stabilizer, or any subset of all of these. It should also be noted that targeted pegylated liposomes can comprise any of the reagents and chemicals discussed herein or known in the art either internal or external to the liposome, including, for example, buffers, salts, and cryoprotectants.
The above method optionally further comprises the step of lyophilizing the composition after the removing step to form a lyophilized composition. As described above, the targeted or non-targeted PTPLA in aqueous solution may comprise a cryoprotectant as described herein or otherwise known in the art. Cryoprotectants may be preferred if the composition is to be lyophilized.
In addition, after the lyophilizing step, the method optionally further comprises the step of reconstituting the lyophilized composition by dissolving the composition in a solvent after the lyophilizing step. Methods of reconstruction are known in the art. One preferred solvent is water. Other preferred solvents include saline solutions and buffered solutions.
While certain exemplary embodiments are discussed herein, it is to be understood that liposomes can be prepared by any method known in the art. See, e.g., g. gregoriadis (ed.), Liposome Technology, volumes 1-3, 1 st edition, 1983; 2 nd edition, 1993, CRC Press,45Boca Raton, Fla. Examples of methods suitable for preparing liposome compositions include extrusion, reverse phase evaporation, sonication, solvent (e.g., ethanol) infusion, microfluidization, detergent dialysis, ether infusion, and dehydration/rehydration. The size of the liposomes can generally be controlled by controlling the pore size of the membrane used for low pressure extrusion or the pressure used in the microfluidics and the number of passes or any other suitable method known in the art.
Typically, α -polyglutamated methotrexate is contained within the interior of the liposome, i.e., the interior (internal) space. In one embodiment, the substituted ammonium is partially or substantially completely removed from the external medium surrounding the liposome. Such removal can be accomplished by any suitable means known in the art (e.g., dilution, ion exchange chromatography, size exclusion chromatography, dialysis, ultrafiltration, and precipitation). Thus, the method of preparing the liposome composition described above or otherwise known in the art may optionally further comprise the step of removing α -polyglutamated methotrexate from an aqueous solution external to the liposomes after the extrusion step.
In other embodiments, the present disclosure provides a targeted pegylated liposomal alpha-polyglutamated methotrexate (PLp-alpha PMTX) that selectively targets the folate receptor, comprising: a liposome comprising an interior space; an alpha-polyglutamated methotrexate disposed within the interior space; a steric stabilizer molecule attached to the exterior of the liposome; and a targeting moiety comprising a protein having specific affinity for at least one folate receptor, said targeting moiety being linked to at least one of a steric stabilizer and the exterior of the liposome. The components of this embodiment may be the same as described for other embodiments of the disclosure. For example, targeted pegylated liposomal α -polyglutamated methotrexate and steric stabilizers that can be PEG are as described in other sections of this disclosure.
In some embodiments, the present disclosure provides a method of making a targeted composition comprising pegylated liposomes comprising entrapped and/or encapsulated alpha polyglutamated methotrexate; the targeting moiety is an amino acid chain comprising a plurality of amino acids, said targeting moiety having a specific affinity for at least one type of folate receptor, said specific affinity being defined as comprised between 0.5x 10 for at least one type of folate receptor -10To 10x 10-6Molar [0.05nM to 10. mu.M]An equilibrium dissociation constant (Kd) within a range, the targeting moiety being attached to one or both of PEG and the exterior of the liposome, the method comprising: forming a mixture comprising a liposome component and alpha-polyglutamated methotrexate in a solution; homogenizing the mixture to form liposomes in the solution; processing the mixture to form liposomes encapsulating and/or encapsulating alpha polyglutamated methotrexate; and embedding and/or encapsulating the alpha-polyglutamated methotrexateThe liposome of (a) provides a targeting moiety on the surface thereof, the targeting moiety having specific affinity for at least one of folate receptor alpha (FR-alpha), folate receptor alpha 0 (FR-alpha 4), and folate receptor (FR-). In some embodiments, the method comprises: forming a mixture comprising a liposome component and α 1 polyglutamated methotrexate in a solution; forming liposomes encapsulating and/or encapsulating the α 2 polyglutamated methotrexate, e.g., by homogenizing or otherwise processing the mixture to form liposomes; and providing a targeting moiety on the surface of the liposome encapsulating and/or encapsulating the α 3 polyglutamated methotrexate, the targeting moiety having specific affinity for at least one of folate receptor α 6(FR- α 7), folate receptor α 5(FR- α 8), and folate receptor (FR-). In some embodiments, the processing comprises one or more of: film hydration, extrusion, in-line mixing, ethanol injection techniques, freeze-thaw techniques, reverse phase evaporation, dynamic high pressure micro-jets, micro-jet mixing, multiple emulsion methods, freeze-dried multiple emulsion methods, 3D printing, membrane contactor methods, and agitation, and once the particles are formed, the size of the particles can be further altered by one or more of extrusion and ultrasonic treatment. In some embodiments, at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75% of the starting material of alpha polyglutamated MTX is encapsulated (embedded) in the targeted liposome during the process of preparing the liposome. In some embodiments, the liposome is anionic or neutral. In some embodiments, the targeting moiety has a specific affinity for one or more of: folate receptor alpha (FR-. alpha.), folate receptor alpha 9 (FR-. beta.), and folate receptor (FR-). In other embodiments, the targeting moiety has specific affinity for folate receptor alpha (FR-alpha) and folate receptor beta (FR-beta). In further embodiments, the targeting moiety has specific affinity for an epitope on a tumor cell surface antigen that is present on a tumor cell but absent or inaccessible on a non-tumor cell.
Liposomes can also be prepared to target specific cells, organs or organelles by altering phospholipid composition or by inserting receptors or counter-receptors into the liposome. For example, liposomes prepared with high levels of nonionic surfactants have been used to target the liver. (see, e.g., Japanese patent 04-244,018 to Hayakawa et al; Kato et al, biol. pharm. Bull.16:960, (1993))) Liposomal formulations of Dipalmitoylphosphatidylcholine (DPPC) with a mixture of soy-derived Sterol Glycosides (SG) and cholesterol (Ch) have also been shown to target the liver. (see Shimizu et al, biol. pharm. Bull.20:881,1997.)
B. Antibody delivery vehicle
In further embodiments, the present disclosure provides an antibody delivery vehicle (e.g., an ADC). In some embodiments, the present disclosure provides an immunoconjugate having the formula (a) - (L) - (α PMTX), wherein: (A) is an antibody or antigen-binding fragment of an antibody; (L) is a linker; and (α PMTX) is an α PMTX composition described herein; and wherein the linker (L) connects (a) to (α PMTX).
In some embodiments, the antibody or antigen-binding antibody fragment has specific affinity for an epitope of a cell surface antigen on a target cell (e.g., an epitope and/or antigen described herein). In certain embodiments, the antibody binds to an antigen target expressed in or on the cell membrane (e.g., on the cell surface) of the cancer/tumor, and upon binding to the (antigen) target, the antibody is internalized by the cell before the α PMTX is released within the cell. In some embodiments, the antibody is a full length antibody.
(A) The antibody or antigen-binding antibody fragment of the- (L) - (α PMTX) immunoconjugate may be an IgA, IgD, IgE, IgG, or IgM antibody. The different classes of immunoglobulins have different and well-known subunit structures and three-dimensional configurations. In certain embodiments, the antibody is an IgG antibody. In some embodiments, the antibody is an IgG1, IgG2, IgG3, or IgG4 antibody. In certain embodiments, the antibody is an IgG1 antibody.
In some embodiments, (a) is an antigen-binding fragment of an antibody. In some embodiments, (a) is an antigen-binding fragment of an antibody.
A "linker" is any chemical moiety capable of linking a compound (typically a drug, such as α PMTX) to an antibody or antigen-binding fragment of an antibody in a stable covalent manner. The linker may be susceptible or substantially resistant to acid-induced cleavage, light-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage under conditions in which the compound or antibody retains activity. Suitable linkers are well known in the art and include, for example, disulfide groups, thioether groups, acid labile groups, photolabile groups, peptidase labile groups, and esterase labile groups. Linkers also include charged linkers and hydrophilic forms thereof.
In some embodiments, the linker is selected from the group consisting of: cleavable linkers, non-cleavable linkers, hydrophilic linkers, and dicarboxylic acid-based linkers. In another embodiment, the linker is a non-cleavable linker. In another embodiment, the linker is selected from the group consisting of: n-succinimidyl 4- (2-pyridyldithio) valerate (SPP); 4- (2-pyridyldithio) butanoic acid N-succinimide ester (SPDB) or 4- (2-pyridyldithio) -2-sulfobutanoic acid N-succinimide ester (sulfo-SPDB); 4- (maleimidomethyl) cyclohexane-carboxylic acid N-succinimidyl ester (SMCC); 4- (maleimidomethyl) cyclohexanecarboxylic acid N-sulfosuccinimidyl ester (sulfo SMCC); n-succinimidyl 4- (iodoacetyl) -aminobenzoate (SIAB); and N-succinimidyl- [ (N-maleimidopropionamido) -tetraethylene glycol ] ester (NHS-PEG 4-maleimide). In another embodiment, the linker is N-succinimidyl- [ (N-maleimido-propionamido) -tetraethylene glycol ] ester (NHS-PEG 4-maleimide).
In some embodiments, the α polyglutamated MTX is linked (coupled) to the antibody or antigen binding antibody fragment of the immunoconjugate, either directly or through a linker, using techniques known in the art. Such attachment of one or more α PMTX may include a number of chemical mechanisms, such as covalent binding, affinity binding, insertion, coordination binding, and complexation. Covalent binding of alpha PMTX to an antibody or antigen-binding antibody fragment can be achieved by direct condensation of existing side chains or by incorporation of external bridging molecules. Many di-or polyvalent agents can be used to associate the polypeptide with other proteins using coupling agents such as carbodiimides, diisocyanates, glutaraldehyde, diazobenzenes, and hexamethylenediamine. This list is not intended to be exhaustive of the various coupling agents known in the art, but rather is exemplary of the more common coupling agents. In some embodiments, the antibody or antigen-binding antibody fragment is derivatized and then linked to α -polyglutamated MTX. Alternatively, α PMTX can be derivatized and linked to an antibody or antigen-binding antibody fragment using techniques known in the art.
In some embodiments, the immunoconjugate comprises an antibody or antigen-binding fragment of an antibody and α PMTX containing 4, 5, 2-10, 4-6, or more than 5 glutamyl groups (including glutamyl groups in methotrexate). In some embodiments, the immunoconjugate comprises alpha polyglutamated methotrexate comprising two or more glutamyl groups in the L form. In other embodiments, the immunoconjugate comprises alpha polyglutamated methotrexate comprising a glutamyl group in D form. In other embodiments, the immunoconjugate comprises alpha polyglutamated methotrexate comprising a glutamyl group in the D form and two or more glutamyl groups in the L form. In further embodiments, the immunoconjugate comprises alpha polyglutamated methotrexate comprising two or more glutamyl groups having a gamma carboxyl linkage. In some embodiments, the immunoconjugate comprises α -pentaglutamated MTX. In other embodiments, the immunoconjugate comprises L-alpha pentaglutamated MTX, D-alpha pentaglutamated MTX, or L-and D-alpha pentaglutamated MTX. In some embodiments, the immunoconjugate comprises α -hexaglutamated MTX (Lp- α PMTX). In other embodiments, the immunoconjugate comprises L- α hexaglutamated MTX, D- α hexaglutamated MTX, or L-and D- α hexaglutamated MTX.
In some embodiments, the antibody delivery vehicle composition comprises alpha polyglutamated methotrexate and an antibody or antigen-binding antibody fragment having specific affinity for an epitope on a cell surface antigen selected from the group consisting of: GONMB, TACTD 2(TROP2), CEACAM5, EPCAM, folate receptor (e.g., folate receptor-alpha, folate receptor-beta, or folate receptor-), mucin 1(MUC-1), MUC-6, STEAP1, mesothelin, bindin 4, ENPP3, Guanylate Cyclase C (GCC), SLC44A4, NaPi2B, CD70(TNFSF7), CA9 (carbonic anhydrase), 5T4(TPBG), SLTRK6, SC-16, tissue factor, LIV-1(ZIP6), CGEN-15027, Pcadherin, fibronectin ectodomain B (ED-B), VEGFR2(CD309), tenascin, collagen IV, periostin, endothelin receptor, HER2, HER 72, EGFR, EGFRvIII, FGFR 72, FGFR3, CD ZD3, CD 36ZD 3, CD3, FGFR3, CD 36ZD 3, CD 36ZD 3, CD, CD19, CD20, CD22, CD26, CD27L, CD28, CD30, CD33, CD34, CD37, CD38, CD40, CD44, CD56, CD70, CD74, CD79, CD79b, CD98, CD105, CD133, CD138, cripto, IGF-1R, IGF-2R, IGF, EphA receptor, EphB receptor, EphA R, IGF, EphB R, IGF, EphA R, IGF, EphB R, IGF, EphUFB R, IGF, EphB R, IGF, integrin (for example, integrin. alpha. v. beta.3,. beta.5 or. alpha. v. beta.6), C antigen, Trk, VEGFR 72, PGR, VEGF-PGR, VEGF-R, IGF, PGRE, PGR, VEGF-R, IGF, PGR, and PG. In some embodiments, the delivery vehicle comprises a targeting moiety with specific affinity for an epitope on a cell surface antigen (such as a neoantigen) derived from or determined to be expressed on a cancer (tumor) in a particular subject.
In some embodiments, the antibody delivery vehicle composition comprises alpha polyglutamated methotrexate and an antibody or antigen-binding antibody fragment having specific affinity for an epitope on an antigen selected from the group consisting of mannose 6-phosphate receptor, transferrin receptor, and Cell Adhesion Molecule (CAM). In other embodiments, the targeting moiety has specific affinity for an epitope of a CAM selected from the group consisting of: intercellular adhesion molecule (ICAM), platelet-endothelial adhesion molecule (PECAM), activated leukocyte adhesion molecule (ALCAM), B-lymphocyte cell adhesion molecule (BL-CAM), Vascular Cell Adhesion Molecule (VCAM), mucosal vascular addressen cell adhesion molecule (MAdCAM), CD44, LFA-2, LFA-3, and basal immunoglobulin.
In some embodiments, the antibody delivery vehicle composition comprises 1, 2, 3, 4, 5-10, or greater than 10 alpha polyglutamated MTX. In some embodiments, the antibody delivery vehicle composition comprises 1, 2, 3, 4, 5-10, or greater than 10 α pentaglutamated MTX. In some embodiments, the antibody delivery vehicle composition comprises 1, 2, 3, 4, 5-10, or greater than 10 α hexaglutamized MTX.
Pharmaceutical compositions and administration
In some embodiments, the liposome composition is provided as a pharmaceutical composition comprising a liposome and a carrier (e.g., a pharmaceutically acceptable carrier). Examples of pharmaceutically acceptable carriers included in the provided pharmaceutical compositions include physiological saline, isotonic dextrose, isotonic sucrose, ringer's solution, and hanks solution. In some embodiments, a buffering substance is added to maintain the optimal pH for storage stability of the pharmaceutical composition. In some embodiments, the pH of the pharmaceutical composition is between 6.0 and 7.5. In some embodiments, the pH is between 6.3 and 7.0. In other embodiments, the pH is 6.5. Ideally, the pH of the pharmaceutical composition allows for the stability of the liposome membrane lipids and retention of the embedded entities. Histidine, hydroxyethylpiperazine-ethylsulfonate (HEPES), Morpholinoethylsulfonate (MES), succinate, tartrate and citrate, typically at concentrations of 2-20mM, are exemplary buffer substances. Other suitable carriers include, for example, water, aqueous buffer, 0.4% NaCl, and 0.3% amino acid. Protein, carbohydrate or polymer stabilizers and tonicity adjusting agents, such as gelatin, albumin, dextran or polyvinylpyrrolidone, may be added. The tonicity of the composition may be adjusted to a physiological level of 0.25-0.35mol/kg with glucose or a more inert compound such as lactose, sucrose, mannitol or dextrin. These compositions may be conventionally sterilized using conventional sterilization techniques known in the art (e.g., by filtration). The resulting aqueous solution may be packaged for use or filtered under sterile conditions and lyophilized, the lyophilized formulation being combined with a sterile aqueous medium prior to administration.
The provided pharmaceutical liposome compositions may also contain other pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, and tonicity adjusting agents, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, and calcium chloride. Additionally, the liposome suspension may include lipid protectors that protect the lipids from free radicals and lipid peroxidation upon storage. Lipophilic free radical quenchers such as alpha tocopherol and water soluble iron specific chelators such as ferrioxamine are suitable.
The concentration of liposomes in the provided fluid pharmaceutical formulations can vary widely as desired, for example, typically less than about 0.05% by weight or at least about 2% -10% by weight up to 30% to 50% by weight and will be selected primarily by fluid volume and viscosity depending on the particular mode of administration selected. For example, the concentration may be increased to reduce the fluid load associated with the treatment. This may be particularly desirable in patients with atherosclerosis-associated congestive heart failure or severe hypertension. Alternatively, the liposomal pharmaceutical composition consisting essentially of the irritating lipid may be diluted to a low concentration to reduce inflammation at the site of administration.
Some embodiments relate to a method of delivering targeted pegylated liposomal formulations of alpha polyglutamated methotrexate to tumors that express folate receptors on the surface. Exemplary methods include the step of administering a liposomal pharmaceutical composition provided herein in an amount to deliver a therapeutically effective dose of targeted pegylated liposomal alpha-polyglutamated methotrexate to a tumor.
The amount of liposomal pharmaceutical composition administered will depend on the particular α -polyglutamated methotrexate entrapped within the liposomes, the disease state being treated, the type of liposomes used, and the judgment of the clinician. Typically, the amount of liposomal pharmaceutical composition administered will be sufficient to deliver a therapeutically effective dose of the particular therapeutic entity.
The amount of liposomal pharmaceutical composition necessary to deliver a therapeutically effective dose can be determined by conventional in vitro and in vivo methods common in the art of pharmaceutical testing. See, e.g., d.b. budman, a.h.calvert, e.k.rowinsky (editors.) Handbook of Anticancer Drug Development, LWW, 2003. Therapeutically effective dosages of various therapeutic compositions are known to those skilled in the art. In some embodiments, the therapeutic entity is delivered via a pharmaceutical liposome composition and provides at least the same or higher activity as that obtained by administering the same amount of therapeutic entity in its conventional non-liposomal formulation. Typically, the dosage of the liposomal pharmaceutical composition most typically ranges between about 0.005 and about 5000mg of the therapeutic entity per square meter of body surface area, between about 0.1 and about 1000mg of the therapeutic entity per square meter of body surface area.
For example, if the subject has a tumor, the effective amount can be an amount of an agent (e.g., an alpha-polyglutamated methotrexate composition) that reduces tumor volume or burden (e.g., as determined by imaging the tumor). The effective amount may also be routinely assessed by the presence and/or frequency of cancer cells in blood or other body fluids or tissues (e.g., biopsies). If the tumor is affecting the normal function of a tissue or organ, the effective amount can be routinely assessed by measuring the normal function of the tissue or organ. In some instances, an effective amount is that amount necessary to alleviate or eliminate one or more, and preferably all, of the symptoms.
Also provided are pharmaceutical compositions comprising alpha polyglutamated methotrexate compositions (e.g., liposomes containing pentaglutamated or hexaglutamated methotrexate). The pharmaceutical composition is a sterile composition comprising liposomes of the sample, and preferably comprising alpha-polyglutamated methotrexate, preferably in a pharmaceutically acceptable carrier.
Unless otherwise indicated herein, a variety of routes of administration may be used. The particular mode selected will depend on the particular active agent selected, the particular condition being treated and the dosage required for therapeutic efficacy. The provided methods may be practiced using any known mode of administration that is medically acceptable and in accordance with good medical practice. In some embodiments, the route of administration is injection. In other embodiments, the injection is by a parenteral route selected from intramuscular, subcutaneous, intravenous, intraarterial, intraperitoneal, intraarticular, intradural, intrathecal, intravenous, intramuscular, or intrasternal injection. In some embodiments, the route of administration is infusion. In additional embodiments, the route of administration is oral, nasal, mucosal, sublingual, intratracheal, ocular, rectal, vaginal, ocular, topical, transdermal, pulmonary, or inhalation.
Therapeutic compositions containing an alpha PMTX composition, e.g., a liposomal alpha PMTX composition as described herein, can be routinely administered intravenously, e.g., by injection of a unit dose. The term "unit dose" when used in relation to the therapeutic compositions provided herein, refers to physically discrete units suitable as unitary dosages for subjects, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a desired diluent (e.g., carrier or vehicle). In a specific embodiment, the therapeutic composition containing the adapter is administered subcutaneously.
In some embodiments, the α -PMTX composition is administered in a manner compatible with dosage formulation and in a therapeutically effective amount. The amount to be administered depends on the subject to be treated, the ability of the subject's system to utilize the active ingredient, and the degree of therapeutic effect desired. The precise amount of active ingredient required for administration depends on the judgment of the practitioner and is specific to each individual. However, suitable dosage ranges for systemic administration are disclosed herein and depend on the route of administration. Suitable regimens for administration are also variable, but are represented by repeated doses at one or more hourly intervals, by initial administration, followed by subsequent injection or other administration. Alternatively, continuous intravenous infusion sufficient to maintain the concentration in the blood within the range prescribed by in vivo therapy is contemplated.
The alpha PMTX compositions are formulated, dosed, and administered in a manner consistent with good medical practice. Considerations in this context include the particular condition being treated, the particular patient being treated, the clinical condition of the individual patient, the etiology of the condition, the site of delivery of the agent, the method of administration, the time course of administration, and other factors known to medical practitioners. The dosage range in which the alpha PMTX composition is administered is that which is large enough to produce the desired effect, wherein the symptoms of the disease mediated by the target molecule are ameliorated. The dose should not be so large as to cause adverse side effects such as high viscosity syndrome, pulmonary edema, congestive heart failure, and other adverse side effects known in the art. In general, the dosage will vary with the age, weight, height, body surface area, health (e.g., kidney and liver function), condition, sex, and extent of the disease of the patient, and can be routinely determined by one of ordinary skill in the art. The dosage may be adjusted by the individual physician if there are any complications.
The dosage schedule and amount effective for therapeutic and prophylactic use (i.e., the "dosing regimen") will depend upon a variety of factors, including the cause, stage and severity of the disease or disorder, the health, physical state, age of the subject being treated, and the site and manner of delivery of the alpha PMTX composition. The therapeutic efficacy and toxicity of the alpha PMTX compositions can be determined by standard pharmaceutical, pharmacological and toxicological procedures in cell culture or experimental animals. The data obtained from these procedures can also be used to formulate a range of human dosages. In addition, the therapeutic index (i.e., the dose therapeutically effective in 50% of the population divided by the dose lethal to 50% of the population (ED50/LD50)) can be readily determined using known procedures. The dosage is preferably within a concentration range that includes the ED50 with little or no toxicity, and may vary within this range depending on the dosage form employed, the sensitivity of the patient, and the route of administration.
The dosage regimen also takes into account pharmacokinetic parameters known in the art, such as drug absorption rate, bioavailability, metabolism, and clearance (see, e.g., Hidalgo-Aragones, J.Steroid biochem. mol. biol.58:611-617 (1996); Groning et al, Pharmazie 51:337-341 (1996); Fotherby, Contraception 54:59-69 (1996); and Johnson et al, J.Pharm. Sci.84:1144-1146 (1995)). For the clinician, determining the dosage regimen for each subject treated is well within the purview of the prior art. In addition, single or multiple doses of the alpha PMTX composition can be administered depending on the dosage and frequency required and tolerated by the subject. The duration of prophylactic and therapeutic treatment will vary depending on the particular disease or condition being treated. Some diseases are amenable to acute treatment, while others require long-term, chronic treatment. The alpha PMTX composition can be administered sequentially or simultaneously with an additional therapeutic agent.
In some embodiments, the alpha PMTX composition is administered in the form of a liposome composition at a dose of between 0.005 and 5000mg of alpha PMTX per square meter of body surface area, or any range therebetween. In other embodiments, the alpha PMTX composition is administered in the form of a liposome composition at a dose of between 0.1 and 1000mg of alpha PMTX per square meter of body surface area, or any range therebetween.
In some embodiments, the alpha PMTX composition is administered in the form of an immunoconjugate composition at a dose of 1mg/kg to 500mg/kg, 1mg/kg to 250mg/kg, 1mg/kg to 200mg/kg, 1mg/kg to 150mg/kg, 1mg/kg to 100mg/kg, 1mg/kg to 50mg/kg, 1mg/kg to 25mg/kg, 1mg/kg to 20mg/kg, 1mg/kg to 15mg/kg, 1mg/kg to 10mg/kg, or 1mg/kg to 5mg/kg, or any range therebetween.
In another embodiment, the alpha PMTX composition is administered in combination with one or more additional therapeutic agents.
In some embodiments, the PLp- α PMTX and/or the targeted PLp- α PMTX is prepared as an infusion composition, an injection composition, a parenteral composition, or a topical composition. In other embodiments, the injection comprises one or more of the following: intraperitoneal injection, direct intratumoral injection, intra-arterial and intravenous injection, subcutaneous injection, intramuscular injection, empirical transdermal and intranasal route delivery. In another embodiment, the PLp- α PMTX and/or the targeted PLp- α PMTX is a liquid solution or suspension. However, also provided herein are solid forms suitable for dissolution or suspension in a liquid vehicle prior to injection. In some embodiments, the targeted pegylated liposomal alpha-polyglutamated methotrexate composition is formulated as an enterically coated tablet or gelcap according to methods known in the art.
In some embodiments, the targeted pegylated liposomal α -polyglutamated methotrexate formulation is administered to a central nervous system tumor directly into the tumor using slow, continuous intracranial infusion of the liposomes (e.g., Convection Enhanced Delivery (CED)). See Saito et al, Cancer Research 64:2572-2579 (2004); mamot et al, J.neuro-Oncology 68:1-9 (2004). In other embodiments, the formulation is applied directly to the tissue surface. Sustained release, pH dependent release, and other specific chemical or environmental condition mediated release delivery of pegylated liposomal alpha-polyglutamated methotrexate formulations (e.g., depot injections and erodible implants) are also provided. Examples of such release-mediated compositions are further described herein or otherwise known in the art.
For administration by inhalation, the compositions may be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant (e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas). In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount.
Where systemic delivery of the compound is desired, the compound may be formulated for parenteral administration by injection (e.g., by bolus injection or continuous infusion). Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers. Parenteral pharmaceutical formulations comprise aqueous solutions of the ingredients. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Alternatively, suspensions of liposomes can be prepared as oil-based suspensions. Suitable lipophilic solvents or vehicles include fatty oils (e.g. sesame oil) or synthetic fatty acid esters (e.g. ethyl oleate or triglycerides).
Alternatively, the non-targeted or targeted pegylated liposomal α -polyglutamated methotrexate can be in powder form or lyophilized form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) prior to use.
The provided compositions (e.g., alpha-polyglutamated methotrexate and liposomes containing alpha-polyglutamated methotrexate) can also be formulated in rectal or vaginal compositions, such as, for example, suppositories or retention enemas containing conventional suppository bases such as cocoa butter or other glycerides.
Methods of use and treatment
In additional embodiments, the present disclosure provides methods of using the alpha polyglutamated methotrexate (alpha PMTX) compositions. In some embodiments, the α α PMTX compositions are used to treat a disease or disorder.
In some embodiments, the present disclosure provides a method of killing a cell, the method comprising contacting the cell with a composition comprising alpha polyglutamated methotrexate (e.g., alpha PMTX disclosed herein). In some embodiments, the contacted cell is a mammalian cell. In other embodiments, the contacted cell is a human cell. In some embodiments, the contacted cell is a hyperproliferative cell. In other embodiments, the hyperproliferative cell is a cancer cell. In other embodiments, the cancer cell is a primary cell or a cell from a cell line obtained/derived from a cancer selected from the group consisting of: non-hematologic malignancies including, for example, lung cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer, bile duct cancer, gall bladder cancer, sarcomas (e.g., osteosarcoma), brain cancer, central nervous system cancer, and melanoma; and hematological malignancies such as leukemia, lymphoma and other B cell malignancies, myeloma and other plasma cell dysplasias or dyscrasias. In other embodiments, the cancer cell is a primary cell or a cell from a cell line obtained/derived from a cancer selected from: breast cancer, head and neck cancer, lung cancer, gastric cancer, osteosarcoma, non-hodgkin lymphoma (NHL), Acute Lymphoblastic Leukemia (ALL), mycosis fungoides (cutaneous T-cell lymphoma), choriocarcinoma and chorioadenoma, non-leukemic meningeal cancer, soft tissue sarcoma (desmoid, invasive fibromatosis), bladder cancer, and Central Nervous System (CNS) lymphoma. In some embodiments, the method is performed in vivo. In other embodiments, the method is performed in vitro. In some embodiments, the alpha PMTX composition contains 4, 5, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the alpha PMTX composition comprises alpha pentaglutamated methotrexate. In some embodiments, the alpha PMTX composition comprises alpha hexaglutaminated methotrexate. In some embodiments, the alpha PMTX composition comprises laa polyglutamated methotrexate. In some embodiments, the alpha PMTX composition comprises D alpha polyglutamated methotrexate. In some embodiments, the alpha PMTX composition comprises L and D alpha polyglutamated methotrexate.
In additional embodiments, the disclosure provides a method of killing a cell, the method comprising contacting the cell with a liposome containing alpha polyglutamated methotrexate (e.g., an Lp-alpha PMTX disclosed herein, such as PLp-alpha PMTX, NTLp-alpha PMTX, NTPLp-alpha PMTX, TLp-alpha PMTX, or TPLp-alpha PMTX). In some embodiments, the liposome is pegylated (e.g., PLp- α PMTX and NTPLp- α PMTX). In some embodiments, the liposomes comprise on their surface a targeting moiety (e.g., TLp-alpha PMTX and TPLp-alpha PMTX) having specific affinity for an epitope of an antigen on the cell surface. In other embodiments, the liposomes are pegylated and comprise on their surface a targeting moiety that specifically binds to an antigen on the cell surface (e.g., TPLp- α PMTX). In some embodiments, the contacted cell is a mammalian cell. In other embodiments, the contacted cell is a human cell. In further embodiments, the contacted cell is a hyperproliferative cell. In other embodiments, the hyperproliferative cell is a cancer cell. In other embodiments, the contacted cancer cell is a primary cell or a cell from a cell line obtained/derived from a cancer selected from the group consisting of: lung cancer (e.g., non-small cell lung cancer), pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer, bile duct cancer, gall bladder cancer, sarcoma (e.g., osteosarcoma), brain cancer, central nervous system cancer, melanoma, myeloma, leukemia, and lymphoma. In some embodiments, the method is performed in vivo. In other embodiments, the method is performed in vitro. In some embodiments, the liposome contains α PMTX that contains 4, 5, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the liposome comprises L α polyglutamated methotrexate. In some embodiments, the liposome comprises D α polyglutamated methotrexate. In some embodiments, the liposome comprises L and D α polyglutamated methotrexate.
In some embodiments, the present disclosure provides a method of killing a hyperproliferative cell, the method comprising contacting the hyperproliferative cell with a delivery vehicle (e.g., a liposome or an antibody) comprising alpha polyglutamated methotrexate (e.g., alpha PMTX disclosed herein). In some embodiments, the delivery vehicle is an antibody (e.g., a full-length IgG antibody, a bispecific antibody, or an scFv). In some embodiments, the delivery vehicle is a liposome (e.g., Lp- α PMTX, such as PLp- α PMTX, NTLp- α PMTX, NTPLp- α PMTX, TLp- α PMTX, or TPLp- α PMTX). In some embodiments, the delivery vehicle is non-targeted. In other embodiments, the delivery vehicle is targeted and comprises on its surface a targeting moiety that has a specific affinity for an epitope on an antigen on the surface of a hyperproliferative cell. In other embodiments, the delivery vehicle comprises a targeting moiety having specific affinity for an epitope on an antigen on the surface of a hyperproliferative cell selected from the group consisting of: GONMB, TACTD 2(TROP2), CEACAM5, EPCAM, folate receptor (e.g., folate receptor-alpha, folate receptor-beta, or folate receptor-), mucin 1(MUC-1), MUC-6, STEAP1, mesothelin, bindin 4, ENPP3, Guanylate Cyclase C (GCC), SLC44A4, NaPi2B, CD70(TNFSF7), CA9 (carbonic anhydrase), 5T4(TPBG), SLTRK6, SC-16, tissue factor, LIV-1(ZIP6), CGEN-15027, Pcadherin, fibronectin ectodomain B (ED-B), VEGFR2(CD309), tenascin, collagen IV, periostin, endothelin receptor, HER2, HER 72, EGFR, EGFRvIII, FGFR 72, FGFR3, CD ZD3, CD 36ZD 3, CD3, FGFR3, CD 36ZD 3, CD 36ZD 3, CD, CD19, CD20, CD22, CD26, CD27L, CD28, CD30, CD33, CD34, CD37, CD38, CD40, CD44, CD56, CD70, CD74, CD79, CD79b, CD98, CD105, CD133, CD138, cripto, IGF-1R, IGF-2R, IGF, EphA receptor, EphB receptor, EphA R, IGF, EphB R, IGF, EphA R, IGF, EphB R, IGF, EphUFB R, IGF, EphB R, IGF, integrin (for example, integrin. alpha. v. beta.3,. beta.5 or. alpha. v. beta.6), C antigen, Trk, VEGFR 72, PGR, VEGF-PGR, VEGF-R, IGF, PGRE, PGR, VEGF-R, IGF, PGR, and PG. In some embodiments, the delivery vehicle comprises a targeting moiety that specifically binds to one or more cell surface antigens (such as neoantigens) derived from or determined to be expressed on a cancer (tumor) in a particular subject. In some embodiments, the method is performed in vivo. In some embodiments, the method is performed in vitro. In some embodiments, the delivery vehicle comprises an alpha PMTX consisting of 4, 5, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the delivery vehicle comprises alpha pentaglutaminated methotrexate. In other embodiments, the delivery vehicle comprises alpha-hexaglutaminated methotrexate. In some embodiments, the delivery vehicle comprises L α polyglutamated methotrexate. In some embodiments, the delivery vehicle comprises D α polyglutamated methotrexate. In some embodiments, the delivery vehicle comprises L and D α polyglutamated methotrexate.
In particular embodiments, the method of killing hyperproliferative cells is performed using a liposomal delivery vehicle comprising alpha polyglutamated methotrexate (e.g., Lp-alpha PMTX disclosed herein, e.g., PLp-alpha PMTX, NTLp-alpha PMTX, NTPLp-alpha PMTX, TLp-alpha PMTX, or TPLp-alpha PMTX). In some embodiments, the delivery vehicle is a non-targeted liposome. In some embodiments, the delivery vehicle comprises on its surface a targeting moiety (e.g., TLp-alpha PMTX and TPLp-alpha PMTX) having specific affinity for an epitope on an antigen on the surface of a hyperproliferative cell. In some embodiments, the delivery vehicle is a liposome comprising on its surface a targeting moiety with specific affinity for an epitope on an antigen on the surface of a hyperproliferative cell. In other embodiments, the targeting moiety has specific affinity for an epitope on an antigen selected from the group consisting of: GONMB, TACTD 2(TROP2), CEACAM5, EPCAM, folate receptor (e.g., folate receptor-alpha, folate receptor-beta, or folate receptor-), mucin 1(MUC-1), MUC-6, STEAP1, mesothelin, bindin 4, ENPP3, Guanylate Cyclase C (GCC), SLC44A4, NaPi2B, CD70(TNFSF7), CA9 (carbonic anhydrase), 5T4(TPBG), SLTRK6, SC-16, tissue factor, LIV-1(ZIP6), CGEN-15027, Pcadherin, fibronectin ectodomain B (ED-B), VEGFR2(CD309), tenascin, collagen IV, periostin, endothelin receptor, HER2, HER 72, EGFR, EGFRvIII, FGFR 72, 3, FGFR3, CD 36ZD 3, CD 36ZD 3, CD 36, CD19, CD20, CD22, CD26, CD27L, CD28, CD30, CD33, CD34, CD37, CD38, CD40, CD44, CD56, CD70, CD74, CD79, CD79b, CD98, CD105, CD133, CD138, cripto, IGF-1R, IGF-2R, IGF, EphA receptor, EphB receptor, EphA R, IGF, EphB R, IGF, EphA R, IGF, EphB R, IGF, EphUFB R, IGF, EphB R, IGF, integrin (for example, integrin. alpha. v. beta.3,. beta.5 or. alpha. v. beta.6), C antigen, Trk, VEGFR 72, PGR, VEGF-PGR, VEGF-R, IGF, PGRE, PGR, VEGF-R, IGF, PGR, and PG. In some embodiments, the liposome comprises a targeting moiety that specifically binds to one or more cell surface antigens (such as neoantigens) derived from or determined to be expressed on a cancer (tumor) in a particular subject. In some embodiments, the liposome is pegylated (e.g., PLp- α PMTX and NTPLp- α PMTX). In other embodiments, the liposomes are pegylated and comprise on their surface a targeting moiety (e.g., TPLp- α PMTX) having specific affinity for an epitope on an antigen on the surface of a hyperproliferative cell. In other embodiments, the liposome is non-pegylated. In some embodiments, the liposomes are non-pegylated and the liposomes comprise on their surface a targeting moiety (e.g., TPLp-alpha PMTX) having specific affinity for an epitope on an antigen on the surface of a hyperproliferative cell. In some embodiments, the liposome comprises an alpha PMTX consisting of 4, 5, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the liposome comprises alpha-tetraglutamated methotrexate. In some embodiments, the liposome comprises alpha pentaglutaminated methotrexate. In other embodiments, the liposome comprises alpha-hexaglutaminated methotrexate. In some embodiments, the liposome comprises L α polyglutamated methotrexate. In some embodiments, the liposome comprises D α polyglutamated methotrexate. In some embodiments, the liposome comprises L and D α polyglutamated methotrexate.
In additional embodiments, the present disclosure provides a method of inhibiting proliferation of a cancer cell, the method comprising contacting the cancer cell with a delivery vehicle (e.g., a liposome or an antibody) comprising alpha polyglutamated methotrexate (e.g., alpha PMTX disclosed herein). In some embodiments, the delivery vehicle is an antibody (e.g., a full-length IgG antibody, a bispecific antibody, or an scFv). In some embodiments, the delivery vehicle is a liposome (e.g., Lp- α PMTX, such as PLp- α PMTX, NTLp- α PMTX, NTPLp- α PMTX, TLp- α PMTX, or TPLp- α PMTX). In some embodiments, the delivery vehicle is non-targeted. In some embodiments, the delivery vehicle is targeted and comprises on its surface a targeting moiety having specific affinity for an epitope on an antigen on the surface of a cancer cell. In other embodiments, the delivery vehicle comprises a targeting moiety having specific affinity for an epitope on a cell surface antigen selected from the group consisting of: GONMB, TACTD 2(TROP2), CEACAM5, EPCAM, folate receptor (e.g., folate receptor-alpha, folate receptor-beta, or folate receptor-), mucin 1(MUC-1), MUC-6, STEAP1, mesothelin, bindin 4, ENPP3, Guanylate Cyclase C (GCC), SLC44A4, NaPi2B, CD70(TNFSF7), CA9 (carbonic anhydrase), 5T4(TPBG), SLTRK6, SC-16, tissue factor, LIV-1(ZIP6), CGEN-15027, Pcadherin, fibronectin ectodomain B (ED-B), VEGFR2(CD309), tenascin, collagen IV, periostin, endothelin receptor, HER2, HER 72, EGFR, EGFRvIII, FGFR 72, FGFR3, CD ZD3, CD 36ZD 3, CD3, FGFR3, CD 36ZD 3, CD 36ZD 3, CD, CD19, CD20, CD22, CD26, CD27L, CD28, CD30, CD33, CD34, CD37, CD38, CD40, CD44, CD56, CD70, CD74, CD79, CD79b, CD98, CD105, CD133, CD138, cripto, IGF-1R, IGF-2R, IGF, EphA receptor, EphB receptor, EphA R, IGF, EphB R, IGF, EphA R, IGF, EphB R, IGF, EphUFB R, IGF, EphB R, IGF, integrin (for example, integrin. alpha. v. beta.3,. beta.5 or. alpha. v. beta.6), C antigen, Trk, VEGFR 72, PGR, VEGF-PGR, VEGF-R, IGF, PGRE, PGR, VEGF-R, IGF, PGR, and PG. In some embodiments, the delivery vehicle is an antibody having specific affinity for an epitope on an antigen on the surface of a cancer cell. In some embodiments, the contacted cancer cell is a mammalian cell. In other embodiments, the contacted cancer cell is a human cell. In further embodiments, the contacted cancer cell is a primary cell or a cell from a cell line obtained/derived from a cancer selected from the group consisting of: lung cancer (e.g., non-small cell lung cancer), pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer, bile duct cancer, gall bladder cancer, sarcoma (e.g., osteosarcoma), brain cancer, central nervous system cancer, melanoma, myeloma, leukemia, and lymphoma. In some embodiments, the method is performed in vivo. In some embodiments, the method is performed in vitro. In some embodiments, the delivery vehicle is an antibody having specific affinity for an epitope on one of the cell surface antigens listed above. In other embodiments, the targeting vehicle is a liposome comprising a targeting moiety having specific affinity for an epitope on the surface of a cancer cell. In other embodiments, the targeting vehicle is a liposome comprising a targeting moiety having specific affinity for an epitope on one of the cell surface antigens listed above. In some embodiments, the delivery vehicle is a pegylated liposome. In other embodiments, the delivery vehicle is a non-pegylated liposome. In some embodiments, the delivery vehicle comprises an alpha PMTX composition containing 4, 5, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the delivery vehicle comprises alpha pentaglutaminated methotrexate. In other embodiments, the delivery vehicle comprises alpha-hexaglutaminated methotrexate. In some embodiments, the delivery vehicle comprises L α polyglutamated methotrexate. In some embodiments, the delivery vehicle comprises D α polyglutamated methotrexate. In some embodiments, the delivery vehicle comprises L and D α polyglutamated methotrexate.
In other embodiments, the present disclosure provides a method of inhibiting proliferation of a cancer cell, the method comprising contacting the cancer cell with a liposome comprising alpha polyglutamated methotrexate (e.g., alpha PMTX disclosed herein). In some embodiments, the liposome is non-targeted. In some embodiments, the liposome is targeted and comprises on its surface a targeting moiety having specific affinity for an epitope on an antigen on the surface of a cancer cell. In other embodiments, the liposome comprises a targeting moiety having specific affinity for an epitope on a cell surface antigen selected from the group consisting of: GONMB, TACTD 2(TROP2), CEACAM5, EPCAM, folate receptor (e.g., folate receptor-alpha, folate receptor-beta, or folate receptor-), mucin 1(MUC-1), MUC-6, STEAP1, mesothelin, bindin 4, ENPP3, Guanylate Cyclase C (GCC), SLC44A4, NaPi2B, CD70(TNFSF7), CA9 (carbonic anhydrase), 5T4(TPBG), SLTRK6, SC-16, tissue factor, LIV-1(ZIP6), CGEN-15027, Pcadherin, fibronectin ectodomain B (ED-B), VEGFR2(CD309), tenascin, collagen IV, periostin, endothelin receptor, HER2, HER 72, EGFR, EGFRvIII, FGFR 72, 3, FGFR3, CD 36ZD 3, CD 36ZD 3, CD 36, CD19, CD20, CD22, CD26, CD27L, CD28, CD30, CD33, CD34, CD37, CD38, CD40, CD44, CD56, CD70, CD74, CD79, CD79b, CD98, CD105, CD133, CD138, cripto, IGF-1R, IGF-2R, IGF, EphA receptor, EphB receptor, EphA R, IGF, EphB R, IGF, EphA R, IGF, EphB R, IGF, EphUFB R, IGF, EphB R, IGF, integrin (for example, integrin. alpha. v. beta.3,. beta.5 or. alpha. v. beta.6), C antigen, Trk, VEGFR 72, PGR, VEGF-PGR, VEGF-R, IGF, PGRE, PGR, VEGF-R, IGF, PGR, and PG. In some embodiments, the delivery vehicle comprises a targeting moiety that specifically binds to one or more cell surface antigens (such as neoantigens) derived from or determined to be expressed on a cancer (tumor) in a particular subject. In some embodiments, the contacted cancer cell is a mammalian cell. In other embodiments, the contacted cancer cell is a human cell. In further embodiments, the contacted cancer cell is a primary cell or a cell from a cell line obtained/derived from a cancer selected from the group consisting of: lung cancer (e.g., non-small cell lung cancer), pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer, bile duct cancer, gall bladder cancer, sarcoma (e.g., osteosarcoma), brain cancer, central nervous system cancer, melanoma, myeloma, leukemia, and lymphoma. In some embodiments, the method is performed in vivo. In some embodiments, the method is performed in vitro. In other embodiments, the targeting vehicle is a liposome comprising a targeting moiety having specific affinity for an epitope on one of the cell surface antigens listed above. In some embodiments, the liposome is pegylated. In other embodiments, the liposome is non-pegylated. In some embodiments, the liposome comprises an alpha PMTX composition containing 4, 5, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the liposome comprises alpha-tetraglutamated methotrexate. In some embodiments, the liposome comprises alpha pentaglutaminated methotrexate. In other embodiments, the liposome comprises alpha-hexaglutaminated methotrexate. In some embodiments, the liposome comprises L α polyglutamated methotrexate. In some embodiments, the liposome comprises D α polyglutamated methotrexate. In some embodiments, the liposome comprises L and D α polyglutamated methotrexate.
In additional embodiments, the present disclosure provides a method for treating a hyperproliferative disorder, comprising administering to a subject having or at risk of having a hyperproliferative disorder an effective amount of a delivery vehicle (e.g., an antibody or liposome) comprising alpha polyglutamated methotrexate (e.g., alpha PMTX disclosed herein). In some embodiments, the delivery vehicle is an antibody (e.g., a full-length IgG antibody, a bispecific antibody, or an scFv). In some embodiments, the delivery vehicle is a liposome (e.g., Lp- α PMTX, such as PLp- α PMTX, NTLp- α PMTX, NTPLp- α PMTX, TLp- α PMTX, or TPLp- α PMTX). In some embodiments, the administered delivery vehicle is pegylated. In some embodiments, the administered delivery vehicle is non-pegylated. In further embodiments, the administered delivery vehicle comprises a targeting moiety with specific affinity for an epitope of an antigen on the surface of the hyperproliferative cell. In further embodiments, the delivery vehicle comprises a targeting moiety having specific affinity for an epitope on a cell surface antigen selected from the group consisting of: GONMB, TACTD 2(TROP2), CEACAM5, EPCAM, folate receptor (e.g., folate receptor-alpha, folate receptor-beta, or folate receptor-), mucin 1(MUC-1), MUC-6, STEAP1, mesothelin, bindin 4, ENPP3, Guanylate Cyclase C (GCC), SLC44A4, NaPi2B, CD70(TNFSF7), CA9 (carbonic anhydrase), 5T4(TPBG), SLTRK6, SC-16, tissue factor, LIV-1(ZIP6), CGEN-15027, Pcadherin, fibronectin ectodomain B (ED-B), VEGFR2(CD309), tenascin, collagen IV, periostin, endothelin receptor, HER2, HER 72, EGFR, EGFRvIII, FGFR 72, 3, FGFR3, CD 36ZD 3, CD 36ZD 3, CD 36, CD19, CD20, CD22, CD26, CD27L, CD28, CD30, CD33, CD34, CD37, CD38, CD40, CD44, CD56, CD70, CD74, CD79, CD79b, CD98, CD105, CD133, CD138, cripto, IGF-1R, IGF-2R, IGF, EphA receptor, EphB receptor, EphA R, IGF, EphB R, IGF, EphA R, IGF, EphB R, IGF, EphUFB R, IGF, EphB R, IGF, integrin (for example, integrin. alpha. v. beta.3,. beta.5 or. alpha. v. beta.6), C antigen, Trk, VEGFR 72, PGR, VEGF-PGR, VEGF-R, IGF, PGRE, PGR, VEGF-R, IGF, PGR, and PG. In some embodiments, the delivery vehicle comprises a targeting moiety that specifically binds to (i.e., has specific affinity for) an epitope on one or more cell surface antigens (such as neoantigens) that are derived from or determined to be expressed on a cancer (tumor) in a particular subject. In some embodiments, the targeting moiety is an antibody or antigen binding antibody fragment. In some embodiments, the administered delivery vehicle is free of a targeting moiety having specific affinity for an epitope on a cell surface antigen of the hyperproliferative cell. In some embodiments, the administered delivery vehicle comprises alpha PMTX containing 4, 5, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the administered delivery vehicle comprises alpha-pentaglutaminated methotrexate. In other embodiments, the administered delivery vehicle comprises alpha-hexaglutaminated methotrexate. In some embodiments, the administered delivery vehicle comprises L α polyglutamated methotrexate. In some embodiments, the administered delivery vehicle comprises D α polyglutamated methotrexate. In some embodiments, the administered delivery vehicle comprises L and D α polyglutamated methotrexate. In some embodiments, the hyperproliferative disorder is cancer. In some embodiments, the hyperproliferative disorder is an autoimmune disease (e.g., rheumatoid arthritis). In some embodiments, the hyperproliferative disorder is a benign or malignant tumor; leukemia, malignant diseases of the blood system or lymphatic system. In other embodiments, the hyperproliferative disorder is selected from the group consisting of: neuronal disorders, glial disorders, astrocytic disorders, hypothalamic disorders, glandular disorders, macrophage disorders, epithelial disorders, mesenchymal disorders, blastocoel disorders, inflammatory disorders, angiogenesis and immune disorders, including autoimmune diseases.
In additional embodiments, the disclosure provides a method for treating a hyperproliferative disorder, comprising administering to a subject having or at risk of having a hyperproliferative disorder an effective amount of liposomes comprising alpha polyglutamated methotrexate (e.g., Lp-alpha PMTX, such as PLp-alpha PMTX, NTLp-alpha PMTX, NTPLp-alpha PMTX, TLp-alpha PMTX, or TPLp-alpha PMTX). In some embodiments, the liposome is pegylated. In some embodiments, the liposome is non-pegylated. In additional embodiments, the liposome comprises a targeting moiety having specific affinity for an epitope of an antigen on the surface of a hyperproliferative cell. In further embodiments, the liposome comprises a targeting moiety having specific affinity for an epitope on a cell surface antigen selected from the group consisting of: GONMB, TACTD 2(TROP2), CEACAM5, EPCAM, folate receptor (e.g., folate receptor-alpha, folate receptor-beta, or folate receptor-), mucin 1(MUC-1), MUC-6, STEAP1, mesothelin, bindin 4, ENPP3, Guanylate Cyclase C (GCC), SLC44A4, NaPi2B, CD70(TNFSF7), CA9 (carbonic anhydrase), 5T4(TPBG), SLTRK6, SC-16, tissue factor, LIV-1(ZIP6), CGEN-15027, Pcadherin, fibronectin ectodomain B (ED-B), VEGFR2(CD309), tenascin, collagen IV, periostin, endothelin receptor, HER2, HER 72, EGFR, EGFRvIII, FGFR 72, 3, FGFR3, CD 36ZD 3, CD 36ZD 3, CD 36, CD19, CD20, CD22, CD26, CD27L, CD28, CD30, CD33, CD34, CD37, CD38, CD40, CD44, CD56, CD70, CD74, CD79, CD79b, CD98, CD105, CD133, CD138, cripto, IGF-1R, IGF-2R, IGF, EphA receptor, EphB receptor, EphA R, IGF, EphB R, IGF, EphA R, IGF, EphB R, IGF, EphUFB R, IGF, EphB R, IGF, integrin (for example, integrin. alpha. v. beta.3,. beta.5 or. alpha. v. beta.6), C antigen, Trk, VEGFR 72, PGR, VEGF-PGR, VEGF-R, IGF, PGRE, PGR, VEGF-R, IGF, PGR, and PG. In some embodiments, the liposomes comprise a targeting moiety having specific affinity for an epitope on one or more cell surface antigens (such as neoantigens) derived from or determined to be expressed on a cancer (tumor) in a particular subject. In some embodiments, the targeting moiety is an antibody or antigen binding antibody fragment. In some embodiments, the liposome is free of a targeting moiety having specific affinity for an epitope on a cell surface antigen of a hyperproliferative cell. In some embodiments, the liposome comprises alpha PMTX containing 4, 5, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the liposome comprises alpha-tetraglutamated methotrexate. In some embodiments, the liposome comprises alpha pentaglutaminated methotrexate. In other embodiments, the liposome comprises alpha-hexaglutaminated methotrexate. In some embodiments, the liposome comprises L α polyglutamated methotrexate. In some embodiments, the liposome comprises D α polyglutamated methotrexate. In some embodiments, the liposome comprises L and D α polyglutamated methotrexate. In some embodiments, the hyperproliferative disorder is cancer. In some embodiments, the hyperproliferative disorder is an autoimmune disease (e.g., rheumatoid arthritis). In some embodiments, the hyperproliferative disorder is a benign or malignant tumor; leukemia, malignant diseases of the blood system or lymphatic system. In other embodiments, the hyperproliferative disorder is selected from the group consisting of: neuronal disorders, glial disorders, astrocytic disorders, hypothalamic disorders, glandular disorders, macrophage disorders, epithelial disorders, mesenchymal disorders, blastocoel disorders, inflammatory disorders, angiogenesis and immune disorders, including autoimmune diseases.
Exemplary hyperproliferative disorders that can be treated according to the disclosed methods include, but are not limited to, disorders associated with benign, premalignant, and malignant cell proliferation, including, but not limited to, neoplasms and tumors (e.g., histiocytoma, glioma, astrocytoma, osteoma), cancer (e.g., lung cancer, small cell lung cancer, gastrointestinal cancer, intestinal cancer, colorectal cancer, breast cancer, ovarian cancer, prostate cancer, testicular cancer, liver cancer, kidney cancer, bladder cancer, pancreatic cancer, brain cancer, sarcomas (e.g., osteosarcoma, kaposi's sarcoma), and melanoma), leukemia, psoriasis, bone disease, fibroproliferative disorders (e.g., fibroproliferative disorders of connective tissue), and atherosclerosis.
In additional embodiments, the present disclosure provides a method for treating cancer, the method comprising administering to a subject having or at risk of having cancer an effective amount of a delivery vehicle (e.g., an antibody or liposome) comprising alpha polyglutamated methotrexate (e.g., alpha PMTX disclosed herein). In some embodiments, the delivery vehicle is an antibody (e.g., a full-length IgG antibody, a bispecific antibody, or an scFv). In some embodiments, the delivery vehicle is a liposome (e.g., Lp- α PMTX, such as PLp- α PMTX, NTLp- α PMTX, NTPLp- α PMTX, TLp- α PMTX, or TPLp- α PMTX). In some embodiments, the administered delivery vehicle is pegylated. In some embodiments, the administered delivery vehicle is non-pegylated. In further embodiments, the administered delivery vehicle comprises a targeting moiety having specific affinity for an epitope of an antigen on the surface of the cancer cell. In further embodiments, the delivery vehicle comprises a targeting moiety having specific affinity for an epitope on a cell surface antigen selected from the group consisting of: GONMB, TACTD 2(TROP2), CEACAM5, EPCAM, folate receptor (e.g., folate receptor-alpha, folate receptor-beta, or folate receptor-), mucin 1(MUC-1), MUC-6, STEAP1, mesothelin, bindin 4, ENPP3, Guanylate Cyclase C (GCC), SLC44A4, NaPi2B, CD70(TNFSF7), CA9 (carbonic anhydrase), 5T4(TPBG), SLTRK6, SC-16, tissue factor, LIV-1(ZIP6), CGEN-15027, Pcadherin, fibronectin ectodomain B (ED-B), VEGFR2(CD309), tenascin, collagen IV, periostin, endothelin receptor, HER2, HER 72, EGFR, EGFRvIII, FGFR 72, FGFR3, CD ZD3, CD 36ZD 3, CD3, FGFR3, CD 36ZD 3, CD 36ZD 3, CD, CD19, CD20, CD22, CD26, CD27L, CD28, CD30, CD33, CD34, CD37, CD38, CD40, CD44, CD56, CD70, CD74, CD79, CD79b, CD98, CD105, CD133, CD138, cripto, IGF-1R, IGF-2R, IGF, EphA receptor, EphB receptor, EphA R, IGF, EphB R, IGF, EphA R, IGF, EphB R, IGF, EphUFB R, IGF, EphB R, IGF, integrin (for example, integrin. alpha. v. beta.3,. beta.5 or. alpha. v. beta.6), C antigen, Trk, VEGFR 72, PGR, VEGF-PGR, VEGF-R, IGF, PGRE, PGR, VEGF-R, IGF, PGR, and PG. In some embodiments, the delivery vehicle comprises a targeting moiety with specific affinity for an epitope on one or more cell surface antigens (such as neoantigens) derived from or determined to be expressed on a cancer (tumor) in a particular subject. In some embodiments, the targeting moiety is an antibody or antigen binding antibody fragment. In some embodiments, the administered delivery vehicle comprises alpha PMTX containing 4, 5, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the administered delivery vehicle comprises alpha-pentaglutaminated methotrexate. In other embodiments, the administered delivery vehicle comprises alpha-hexaglutaminated methotrexate. In some embodiments, the administered delivery vehicle comprises L α polyglutamated methotrexate. In some embodiments, the administered delivery vehicle comprises D α polyglutamated methotrexate. In some embodiments, the administered delivery vehicle comprises L and D α polyglutamated methotrexate. In some embodiments, the cancer is selected from the group consisting of: lung cancer (e.g., non-small cell lung cancer), pancreatic cancer, breast cancer, ovarian cancer, lung cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colon cancer, esophageal cancer, cervical cancer, renal cancer, bile duct cancer, gallbladder cancer, and hematologic malignancies (e.g., leukemia or lymphoma).
In additional embodiments, the disclosure provides a method for treating cancer, the method comprising administering to a subject having or at risk of having cancer an effective amount of liposomes comprising alpha polyglutamated methotrexate (e.g., Lp-alpha PMTX, such as PLp-alpha PMTX, NTLp-alpha PMTX, NTPLp-alpha PMTX, TLp-alpha PMTX, or TPLp-alpha PMTX). In some embodiments, the liposome is pegylated. In some embodiments, the liposome is non-pegylated. In additional embodiments, the liposome comprises a targeting moiety having specific affinity for an epitope of an antigen on the surface of a cancer cell. In further embodiments, the liposome comprises a targeting moiety having specific affinity for an epitope on a cell surface antigen selected from the group consisting of: GONMB, TACTD 2(TROP2), CEACAM5, EPCAM, folate receptor (e.g., folate receptor-alpha, folate receptor-beta, or folate receptor-), mucin 1(MUC-1), MUC-6, STEAP1, mesothelin, bindin 4, ENPP3, Guanylate Cyclase C (GCC), SLC44A4, NaPi2B, CD70(TNFSF7), CA9 (carbonic anhydrase), 5T4(TPBG), SLTRK6, SC-16, tissue factor, LIV-1(ZIP6), CGEN-15027, Pcadherin, fibronectin ectodomain B (ED-B), VEGFR2(CD309), tenascin, collagen IV, periostin, endothelin receptor, HER2, HER 72, EGFR, EGFRvIII, FGFR 72, FGFR3, CD ZD3, CD 36ZD 3, CD3, FGFR3, CD 36ZD 3, CD 36ZD 3, CD, CD19, CD20, CD22, CD26, CD27L, CD28, CD30, CD33, CD34, CD37, CD38, CD40, CD44, CD56, CD70, CD74, CD79, CD79b, CD98, CD105, CD133, CD138, cripto, IGF-1R, IGF-2R, IGF, EphA receptor, EphB receptor, EphA R, IGF, EphB R, IGF, EphA R, IGF, EphB R, IGF, EphUFB R, IGF, EphB R, IGF, integrin (for example, integrin. alpha. v. beta.3,. beta.5 or. alpha. v. beta.6), C antigen, Trk, VEGFR 72, PGR, VEGF-PGR, VEGF-R, IGF, PGRE, PGR, VEGF-R, IGF, PGR, and PG. In some embodiments, the liposomes comprise a targeting moiety having specific affinity for an epitope on one or more cell surface antigens (such as neoantigens) derived from or determined to be expressed on a cancer (tumor) in a particular subject. In some embodiments, the targeting moiety is an antibody or antigen binding antibody fragment. In some embodiments, the liposome comprises alpha PMTX containing 4, 5, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the liposome comprises alpha-tetraglutamated methotrexate. In some embodiments, the liposome comprises alpha pentaglutaminated methotrexate. In other embodiments, the liposome comprises alpha-hexaglutaminated methotrexate. In some embodiments, the liposome comprises L α polyglutamated methotrexate. In some embodiments, the liposome comprises D α polyglutamated methotrexate. In some embodiments, the liposome comprises L and D α polyglutamated methotrexate. In some embodiments, the cancer is selected from the group consisting of: lung cancer (e.g., non-small cell lung cancer), pancreatic cancer, breast cancer, ovarian cancer, lung cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colon cancer, esophageal cancer, cervical cancer, renal cancer, bile duct cancer, gallbladder cancer, and hematologic malignancies (e.g., leukemia or lymphoma).
In additional embodiments, the present disclosure provides a method for treating cancer, the method comprising administering to a subject having or at risk of having cancer an effective amount of a liposome composition comprising liposomes comprising alpha polyglutamated methotrexate and a targeting moiety having specific affinity for an epitope of an antigen on the surface of the cancer. In some embodiments, the liposome comprises a targeting moiety having specific affinity for an epitope on a cell surface antigen selected from the group consisting of: GONMB, TACTD 2(TROP2), CEACAM5, EPCAM, folate receptor (e.g., folate receptor-alpha, folate receptor-beta, or folate receptor-), mucin 1(MUC-1), MUC-6, STEAP1, mesothelin, bindin 4, ENPP3, Guanylate Cyclase C (GCC), SLC44A4, NaPi2B, CD70(TNFSF7), CA9 (carbonic anhydrase), 5T4(TPBG), SLTRK6, SC-16, tissue factor, LIV-1(ZIP6), CGEN-15027, Pcadherin, fibronectin ectodomain B (ED-B), VEGFR2(CD309), tenascin, collagen IV, periostin, endothelin receptor, HER2, HER 72, EGFR, EGFRvIII, FGFR 72, FGFR3, CD ZD3, CD 36ZD 3, CD3, FGFR3, CD 36ZD 3, CD 36ZD 3, CD, CD19, CD20, CD22, CD26, CD27L, CD28, CD30, CD33, CD34, CD37, CD38, CD40, CD44, CD56, CD70, CD74, CD79, CD79b, CD98, CD105, CD133, CD138, cripto, IGF-1R, IGF-2R, IGF, EphA receptor, EphB receptor, EphA R, IGF, EphB R, IGF, EphA R, IGF, EphB R, IGF, EphUFB R, IGF, EphB R, IGF, integrin (for example, integrin. alpha. v. beta.3,. beta.5 or. alpha. v. beta.6), C antigen, Trk, VEGFR 72, PGR, VEGF-PGR, VEGF-R, IGF, PGRE, PGR, VEGF-R, IGF, PGR, and PG. In some embodiments, the liposomes comprise a targeting moiety having specific affinity for an epitope on one or more cell surface antigens (such as neoantigens) derived from or determined to be expressed on a cancer (tumor) in a particular subject. In some embodiments, the administered liposome composition comprises pegylated liposomes (e.g., TPLp-alpha PMTX). In some embodiments, the administered liposome composition comprises non-pegylated liposomes. In some embodiments, the liposomes of the administered liposome composition comprise alpha PMTX containing 4, 5, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the liposomes of the administered liposome composition comprise alpha-tetraglutamated methotrexate. In some embodiments, the liposomes of the administered liposome composition comprise alpha-pentaglutaminated methotrexate. In other embodiments, the liposomes of the administered liposome composition comprise alpha-hexaglutaminated methotrexate. In some embodiments, the liposome composition is administered to treat a cancer selected from the group consisting of: lung cancer, pancreatic cancer, breast cancer, ovarian cancer, lung cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colon cancer, esophageal cancer, cervical cancer, renal cancer, bile duct cancer, gallbladder cancer, and hematologic malignancies.
In some embodiments, the liposome comprises a targeting moiety having specific affinity for an epitope of a Tumor Specific Antigen (TSA) or Tumor Associated Antigen (TAA). In some embodiments, the liposome comprises a targeting moiety having specific affinity for an epitope of an antigen selected from the group consisting of: tumor differentiation antigens (e.g., MART1/Melana, GP100(Pmel 17), tyrosinase, TRP1 and TRP2), tumor specific multiple lineage antigens (e.g., MAGE1, MAGE3, BAGE, GAGE1, GAGE2 and p15), overexpressed embryonic antigens (e.g., carcinoembryonic antigen (CEA)), overexpressed oncogenes or mutated tumor suppressor gene products (e.g., p53, Ras and HER2/neu), unique tumor antigens resulting from chromosomal translocations (e.g., BCR-ABL, E2A-PRL, H4-RET, IGH-IGK and MYL-RAR), viral antigens (e.g., Epstein Barr virus antigen EBVA, Human Papilloma Virus (HPV) antigen E6 or E7), GP 100), Prostatic Acid Phosphatase (PAP), Prostate Specific Antigen (PSA), PTGER4, CD 4, EBVA 4662, CD52, EVGA 69-CD 4624, CD 465, CXCR 24, CD 39361, CD 3946, CD 24, CD 2I, SLC39A8, MICB, LRRC70, CLELC2B, HMHA1, LST1, and CMTM6 CKLFSF 6).
In some embodiments, the liposome comprises a targeting moiety having specific affinity for an epitope of a hematologic tumor antigen. In other embodiments, the targeting moiety has specific affinity for an epitope of a hematologic tumor antigen selected from the group consisting of: CD19, CD20, CD22, CD30, CD138, CD33 CD34, CD38, CD123, CS1, ROR1, LewisYIg kappa light chain, TCR, BCMA, TACI, BAFFR (CD268), cala, and NKG2DL ligands). In some embodiments, the liposome comprises a targeting moiety having specific affinity for an epitope of a B cell lymphoma specific idiotype immunoglobulin or B cell differentiation antigen (e.g., CD19, CD20, and CD 37). In some embodiments, the liposome comprises a targeting moiety with specific affinity for an epitope of an antigen on multiple myeloma cells (e.g., CS-1, CD38, CD138, MUC1, HM1.24, CYP1B1, SP17, PRAME, wilms tumor 1WT1), and heat shock protein gp96) or on myeloid cells (e.g., TSLPR and IL-7R).
In some embodiments of the present invention, the substrate is,the liposomes comprise a targeting moiety having specific affinity for an epitope of a solid tumor antigen. In other embodiments, the targeting moiety has specific affinity for an epitope of a hematologic tumor antigen selected from the group consisting of: disialoganglioside (GD2), o-acetyl GD2, EGFRvIII, ErbB2, VEGFR2, FAP, mesothelin, IL13Ra2 (glioma), cMET, PSMA, L1CAM, CEA, and EGFR. In some embodiments, the liposome comprises a targeting moiety having specific affinity for an epitope of an antigen selected from the group consisting of: GONMB, TACTD 2(TROP2), CEACAM5, EPCAM, folate receptor (e.g., folate receptor-alpha, folate receptor-beta, or folate receptor-), mucin 1(MUC-1), MUC-6, STEAP1, mesothelin, bindin 4, ENPP3, Guanylate Cyclase C (GCC), SLC44A4, NaPi2B, CD70(TNFSF7), CA9 (carbonic anhydrase), 5T4(TPBG), SLTRK6, SC-16, tissue factor, LIV-1(ZIP6), CGEN-15027, Pcadherin, fibronectin ectodomain B (ED-B), VEGFR2(CD309), tenascin, collagen IV, periostin, endothelin receptor, HER2, HER 72, EGFR, EGFRvIII, FGFR 72, FGFR3, CD ZD3, CD 36ZD 3, CD3, FGFR3, CD 36ZD 3, CD 36ZD 3, CD, CD19, CD20, CD22, CD26, CD27L, CD28, CD30, CD33, CD34, CD37, CD38, CD40, CD44, CD56, CD70, CD74, CD79, CD79b, CD98, CD105, CD133, CD138, cripto, IGF-1R, IGF-2R, IGF, EphA receptor, EphB receptor, EphA R, IGF, EphB R, IGF, EphA R, IGF, EphB R, IGF, EphUFB R, IGF, EphB R, IGF, integrin (for example, integrin. alpha. v. beta.3,. beta.5 or. alpha. v. beta.6), C antigen, Trk, VEGFR 72, PGR, VEGF-PGR, VEGF-R, IGF, PGRE, PGR, VEGF-R, IGF, PGR, and PG. In some embodiments, the liposome comprises a targeting moiety having specific affinity for an epitope of an antigen selected from the group consisting of: CD137, PDL1, CTLA4, CD47, KIR, TNFRSF10B (DR5), T IM3, PD1, cMet, glycolipid F77, EGFRvIII, HLAA2(NY-ESO-1), LAG3, CD134(OX40), HVEM, BTLA, TNFRSF25(DR3), CD133, MAGE A3, PSCA, MUC1, CD44v6, CD44v6/7, CD44v7/8, IL11Ra, ephA2, CAIX, MNCAIX, CSPG4, MUC16, EPCAM (EGP2), TAG72, EGP40, ErbB receptor family, ErbB2(HER2), ErbB3/4, RAGE1, RAGE 3, FAR, LewisYNCAM, HLAA1/MAGE1, MAGEA1, MAGEA3, MAGE-A3, B7H3, WT 3, MelanA (MART 3), HPVE 3, thyroglobulin, tyrosinase, PSA, CLL1GD3, Tn Ag, FLT3, KIT, PRSS 3, CD3, PDGFR-beta, SSEA 3, prostatase (prostase), PAP, ELF 23, ephB 3, IGF 3, IGFII, IGFI receptor, LMP 3, gp100, bcr-ab 3, glycosyl 3, sLe, GM3, TGS 3, folate receptor beta, TEM 3 (CD248 36248 3), TEM7 3, TSN 3, ORF 5 HR, CX3672, CD3, HLAG 3, HLE 3, HLS 3, HALT 72, HALT 23, HALT 72, HALF 3, PGCR 1-3, HAVC 3, HALF 3, HALP-36, hTERT, sarcoma translocation breakpoint, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin B1, MYCN, RhoC, TRP2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP4, SSX2, reverse transcriptase, RU1, RU2, intestinal carboxyesterase, neutrophil elastase, mut hsp70-2, CD79a, CD79B, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12 LF, BST LF, EMR LF, LY LF, NuGPC LF, FCTSP RLS, IGLL LF, BCA-180, MAGE LF, VEGFR 72, GARP 11, IGF 3-7, MAG LF, BCG 7-7, MAG LF, MAG-9-7, MAG-9-7, MAG-9, MAG-7, MAG-LF, MAG-7, MAGE LF, CA 27.29(BCAA), CA195, CA242, CA-50, CAM43, CD68, CO-029, FGF5, G250, HTgp-175, M344, MA50, MG7-Ag, MOV18, NB/70K, NY-CO1, RCAS1, SDCCAG16, M2BP, TAAL6, TLP and TPS, glioma-associated antigen, alpha-feto egg White (AFP), p26 fragments of AFP, lectin-reactive AFP, and TLR 4.
In some embodiments, the liposome comprises a targeting moiety having specific affinity for an epitope of an antigen selected from the group consisting of: PDG, VEGFR, neuropilin 1 (NRP), neuropilin 2 (NRP), betacellulin, PLGF, RET (transfection rearrangement), TIE (TEK), CA125, CD CD, CD32, CD (e.g., CD44 v), CD49 (integrin α 5), CD (ICAM), CD200, CD147, CD166, CD200, ESA, SHH, DHH, IHH, patched 1 (PTCH), Smoothened (SMO), WNT2, WNT3, WNT4, WNT5, WNT7, WNT8, WNT10, EAT 16, LKP, LRP, Dc, FZD, ZD, JAWNT 7, WNT8, TNFRSF (TNFRSF) TNFRSF11, TNFRSF (TNFRSF) TNFRSF, TNFRSF (TNFRSF) and TNFRSF (TNFRSF) ligand (TNFRSF 11, TNFRSF) and TNFRSF, TNFRSF (TROY), TNFRSF (DR), ILIRI, 1L1R, IL2, IL5, IL6, 1L8, IL10, IL12, IL13, IL15, IL18, IL19, IL21, IL23, REGIV, FGFR, ALK, ALCAM, Axl, TGFb, TGFBR, IGFIR, BMPRI, N-cadherin, E-cadherin, VE-cadherin, ganglioside GM, ganglioside GD, PSGR, DCC, CDCP, CXCR, CCR, SEALAN 1, SEALAN 2, SEALAN 3, SEALAN 4, TMF, neuregulin, MCSF, CSF, CSFR (fms), GCSF, SFAM, BCBCCA, HLA-BRDR, SMCC, VLCC, VLCA, LYCA, LACA, CALCR, LIGA, SACCA, SACCS, CALCR, LICA, CALCR, LCR, CAGR, CANG, CAGR, CA, Leukotriene B4 receptor (LTB4R), neurotensin NT receptor (NTR), 5T4 carcinoembryonic antigen, tenascin C, MMP2, MMP7, MMP9, MMP12, MMP14, MMP26, cathepsin G, SULF1, SULF2, MET, CA9, TM4SF1, syndecan (SDCl), ephrin B4, TEM1, TGF β 1 and TGFBRII.
In some embodiments, the liposomes comprise a targeting moiety that has specific affinity for an epitope of an antigen associated with an immune system disorder (e.g., autoimmune and inflammatory disorders), or is associated with modulating an immune response. In some embodiments, the targeting moiety has specific affinity for an epitope of a cell surface antigen expressed on the surface of a macrophage (expressing CD 44).
In some embodiments, the liposome comprises a targeting moiety having specific affinity for an epitope of an immunosuppressive target. In another embodiment, AD is an epitope of an immunosuppressive target selected from the group consisting of: IL1Ra, IL6R, CD26L, CD28, CD80, Fc γ RIIB. In another embodiment, the AD in the adaptor is an epitope of an immunostimulatory target selected from the group consisting of: CD25, CD28, CTLA4, PD1, B7H1(PDL1), B7H4 TGF β, TNFRSF4(OX40), TNFRSF5(CD40), TNFRSF9(41BB, CD137), TNFRSF14(HVEM), TNFRSF25(DR3) and TNFRSF18 (GITR).
In some embodiments, the liposome comprises a targeting moiety having specific affinity for an epitope of an antigen selected from the group consisting of: IL1Rb, C3AR, C5AR, CXCR1, CXCR2, CCR1, CCR3, CCR7, CCR8, CCR9, CCR10, ChemR23, MPL, GP130, TLR2, TLR3, TLR4, TLR5, TLR7, TREM 7, CD49 7 (integrin alpha 1), integrin a5B 7, alpha 4B7 integrin, cathepsin 7 (LTBR), TNFRSF7 (Fas, CD 7), TNFRSF6 7 (DcCSF), TNFRSF7(CD 7), TNFRSF11 7 (RANK), TNFRSF7 (NGFR 7), TNFRSF19 (RELT), TNFRSF7 (TNFRSF 7), TNFRSF7, TNFRFSF 7, TNFRSF 363672, TNFRSF 363636363672, TNFRSF7, TNFRSF 36363636363672, TNFRSF 36363672, TNFRSF7, TNFSK 36363672, TNFRSF 3636363636363672, TNFSK 363672, TNFRSF 363636363636363672, TNFRSF7, TNFRSF 3636363672, TNFRSF7, TNFSK 3636363672, TNFRSF 363636363636363636363636363636363636363672, TNFRSF 36, CD2, CD4, CD11a, CD18, CD30, CD40, CD86, CXCR3, CCR2, CCR4, CCR5, CCR8, RhD, IgE and Rh.
In additional embodiments, the present disclosure provides a method for treating cancer, the method comprising administering to a subject having or at risk of having a cancer that expresses a folate receptor on the cell surface thereof an effective amount of a liposomal composition, wherein the liposomal composition comprises liposomes comprising (a) alpha polyglutamated methotrexate (alpha PMTX) and (b) a targeting moiety having specific binding affinity for a folate receptor. In some embodiments, the targeting moiety has specific binding affinity for folate receptor alpha (FR-alpha), folate receptor beta (FR-beta), and/or folate receptor (FR-). In some embodiments, the targeting moiety has specific binding affinity for folate receptor alpha (FR-alpha), folate receptor beta (FR-beta), and/or folate receptor (FR-). In some embodiments, the targeting moiety has specific binding affinity for folate receptor alpha (FR-alpha) and folate receptor beta (FR-beta). In some embodiments, the administered liposome composition comprises pegylated liposomes (e.g., TPLp-alpha PMTX). In some embodiments, the administered liposome composition comprises non-pegylated liposomes. In some embodiments, the liposomes of the administered liposome composition comprise alpha PMTX containing 4, 5, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the liposomes of the administered liposome composition comprise alpha-tetraglutamated methotrexate. In some embodiments, the liposomes of the administered liposome composition comprise alpha-pentaglutaminated methotrexate. In other embodiments, the liposomes of the administered liposome composition comprise alpha-hexaglutaminated methotrexate. In some embodiments, the liposome composition is administered to treat epithelial tissue malignancies. In some embodiments, the liposome composition is administered to treat a cancer selected from the group consisting of: lung cancer, pancreatic cancer, breast cancer, ovarian cancer, lung cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colon cancer, esophageal cancer, cervical cancer, renal cancer, bile duct cancer, gallbladder cancer, and hematologic malignancies.
In some embodiments, the present disclosure provides a method for treating lung cancer (e.g., non-small cell lung cancer), the method comprising administering to a subject having or at risk of having lung cancer an effective amount of a delivery vehicle (e.g., an antibody or liposome) comprising alpha polyglutamated methotrexate (e.g., alpha PMTX disclosed herein). In certain embodiments, the cancer is non-small cell lung cancer. In some embodiments, the delivery vehicle is an antibody (e.g., a full-length IgG antibody, a bispecific antibody, or an scFv). In some embodiments, the delivery vehicle is a liposome (e.g., Lp- α PMTX, such as PLp- α PMTX, NTLp- α PMTX, NTPLp- α PMTX, TLp- α PMTX, or TPLp- α PMTX). In some embodiments, the administered delivery vehicle is pegylated. In some embodiments, the administered delivery vehicle is non-pegylated. In further embodiments, the delivery vehicle comprises on its surface a targeting moiety having specific affinity for an epitope on an antigen on the surface of a lung cancer (e.g., non-small cell lung cancer) cell. In other embodiments, the delivery vehicle comprises a targeting moiety having specific affinity for an epitope on an antigen selected from the group consisting of: mucin 1, bindin 4, NaPi2b, CD56, EGFR and SC-16. In some embodiments, the targeting moiety is an antibody or a fragment of an antibody. In further embodiments, the delivery vehicle is a liposome and the liposome comprises a targeting moiety having specific affinity for an epitope on an antigen selected from the group consisting of: mucin 1, bindin 4, NaPi2b, CD56, EGFR and SC-16. In other embodiments, the delivery vehicle is a pegylated liposome comprising a targeting moiety having specific affinity for an epitope on an antigen selected from the group consisting of: mucin 1, bindin 4, NaPi2b, CD56, EGFR and SC-16. In some embodiments, the administered delivery vehicle comprises alpha PMTX containing 4, 5, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the administered delivery vehicle comprises alpha-pentaglutaminated methotrexate. In other embodiments, the administered delivery vehicle comprises alpha-hexaglutaminated methotrexate. In some embodiments, the administered delivery vehicle comprises L α polyglutamated methotrexate. In some embodiments, the administered delivery vehicle comprises D α polyglutamated methotrexate. In some embodiments, the administered delivery vehicle comprises L and D α polyglutamated methotrexate.
In some embodiments, the present disclosure provides a method for treating pancreatic cancer, the method comprising administering to a subject having or at risk of having pancreatic cancer an effective amount of a delivery vehicle (e.g., an Antibody (ADC) or liposome) comprising alpha polyglutamated methotrexate (e.g., alpha PMTX disclosed herein). In some embodiments, the delivery vehicle is an antibody (e.g., a full-length IgG antibody, a bispecific antibody, or an scFv). In some embodiments, the delivery vehicle is a liposome (e.g., Lp- α PMTX, such as PLp- α PMTX, NTLp- α PMTX, NTPLp- α PMTX, TLp- α PMTX, or TPLp- α PMTX). In some embodiments, the administered delivery vehicle is pegylated. In some embodiments, the administered delivery vehicle is non-pegylated. In further embodiments, the delivery vehicle comprises on its surface a targeting moiety having specific affinity for an epitope on an antigen on the surface of a pancreatic cancer cell. In other embodiments, the delivery vehicle comprises a targeting moiety having specific affinity for an epitope on an antigen selected from the group consisting of: tactd 2(TROP2), mucin 1, mesothelin, Guanylate Cyclase C (GCC), SLC44a4, and bindin 4. In other embodiments, the delivery vehicle is a liposome and the liposome comprises a targeting moiety having specific affinity for an epitope on an antigen selected from the group consisting of: tactd 2(TROP2), mucin 1, mesothelin, Guanylate Cyclase C (GCC), SLC44a4, and bindin 4. In some embodiments, the administered delivery vehicle comprises alpha PMTX containing 4, 5, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the administered delivery vehicle comprises alpha-pentaglutaminated methotrexate. In other embodiments, the administered delivery vehicle comprises alpha-hexaglutaminated methotrexate. In some embodiments, the administered delivery vehicle comprises L α polyglutamated methotrexate. In some embodiments, the administered delivery vehicle comprises D α polyglutamated methotrexate. In some embodiments, the administered delivery vehicle comprises L and D α polyglutamated methotrexate.
In additional embodiments, the present disclosure provides a method for treating breast cancer (e.g., triple negative breast cancer (estrogen receptor-, progestin receptor-and HER2)) comprising administering to a subject having or at risk of having breast cancer an effective amount of a delivery vehicle (e.g., an antibody or liposome) comprising alpha polyglutamated methotrexate (e.g., alpha PMTX disclosed herein). In some embodiments, the delivery vehicle administered is a liposome comprising alpha-polyglutamated methotrexate. In some embodiments, the delivery vehicle is an antibody (e.g., a full-length IgG antibody, a bispecific antibody, or an scFv). In some embodiments, the delivery vehicle is a liposome (e.g., Lp- α PMTX, such as PLp- α PMTX, NTLp- α PMTX, NTPLp- α PMTX, TLp- α PMTX, or TPLp- α PMTX). In some embodiments, the administered delivery vehicle is pegylated. In some embodiments, the administered delivery vehicle is non-pegylated. In a further embodiment, the delivery vehicle comprises on its surface a targeting moiety having specific affinity for an epitope on an antigen on the surface of a breast cancer cell. In other embodiments, the delivery vehicle comprises a targeting moiety having specific affinity for an epitope on an antigen selected from the group consisting of: LIV-1(ZIP6), EGFR, HER2, HER3, mucin 1, gon mb, and bindin 4. In some embodiments, the targeting moiety is an antibody or a fragment of an antibody. In further embodiments, the delivery vehicle is a liposome and the liposome comprises a targeting moiety having specific affinity for an epitope on an antigen selected from the group consisting of: LIV-1(ZIP6), EGFR, HER2, HER3, mucin 1, gon mb, and bindin 4. In some embodiments, the administered delivery vehicle comprises alpha PMTX containing 4, 5, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the administered delivery vehicle comprises alpha-pentaglutaminated methotrexate. In other embodiments, the administered delivery vehicle comprises alpha-hexaglutaminated methotrexate. In some embodiments, the administered delivery vehicle comprises L α polyglutamated methotrexate. In some embodiments, the administered delivery vehicle comprises D α polyglutamated methotrexate. In some embodiments, the administered delivery vehicle comprises L and D α polyglutamated methotrexate.
In some embodiments, the present disclosure provides a method for treating a hematologic cancer, the method comprising administering to a subject having or at risk of having a hematologic cancer an effective amount of a delivery vehicle (e.g., an antibody or liposome) comprising alpha polyglutamated methotrexate (e.g., alpha PMTX disclosed herein). In some embodiments, the delivery vehicle is an antibody (e.g., a full-length IgG antibody, a bispecific antibody, or an scFv). In some embodiments, the delivery vehicle is a liposome (e.g., Lp- α PMTX, such as PLp- α PMTX, NTLp- α PMTX, NTPLp- α PMTX, TLp- α PMTX, or TPLp- α PMTX). In some embodiments, the administered delivery vehicle is pegylated. In some embodiments, the administered delivery vehicle is non-pegylated. In further embodiments, the delivery vehicle comprises on its surface a targeting moiety having specific affinity for an epitope on an antigen on the surface of a hematologic cancer cell. In other embodiments, the delivery vehicle comprises a targeting moiety having specific affinity for an epitope on an antigen selected from the group consisting of: CD30, CD79b, CD19, CD138, CD74, CD37, CD19, CD22, CD33, CD34, and CD 98. In other embodiments, the delivery vehicle is a liposome and the liposome comprises a targeting moiety having specific affinity for an epitope on an antigen selected from the group consisting of: CD30, CD79b, CD19, CD138, CD74, CD37, CD19, CD22, CD33, CD34, and CD 98. In some embodiments, the administered delivery vehicle comprises alpha PMTX containing 4, 5, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the administered delivery vehicle comprises alpha-pentaglutaminated methotrexate. In other embodiments, the administered delivery vehicle comprises alpha-hexaglutaminated methotrexate. In some embodiments, the administered delivery vehicle comprises L α polyglutamated methotrexate. In some embodiments, the administered delivery vehicle comprises D α polyglutamated methotrexate. In some embodiments, the administered delivery vehicle comprises L and D α polyglutamated methotrexate.
In some embodiments, the present disclosure provides a method for treating a subject having or at risk of having a cancer that is distinguishable by the expression of an antigen on the cell surface thereof. Thus, in some embodiments, the present disclosure provides a method for treating cancer, the method comprising administering to a subject having or at risk of having cancer an effective amount of a delivery vehicle (e.g., an antibody or liposome) comprising a targeting moiety having specific affinity for an epitope on a surface antigen of the cancer and alpha polyglutamated methotrexate (e.g., alpha PMTX disclosed herein). In some embodiments, the administered delivery vehicle is pegylated. In some embodiments, the targeting moiety is an antibody or a fragment of an antibody. In further embodiments, the delivery vehicle is a liposome. In some embodiments, the administered delivery vehicle comprises an alpha PMTX consisting of 4, 5, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the administered delivery vehicle comprises alpha-pentaglutaminated methotrexate. In other embodiments, the administered delivery vehicle comprises alpha-hexaglutaminated methotrexate. In some embodiments, the administered delivery vehicle comprises L α polyglutamated methotrexate. In some embodiments, the administered delivery vehicle comprises D α polyglutamated methotrexate. In some embodiments, the administered delivery vehicle comprises L and D α polyglutamated methotrexate.
In some embodiments, the disclosed compositions (e.g., liposomes containing alpha-polyglutamated methotrexate) are administered to a subject having, or at risk of having, a cancer, a solid tumor and/or a metastasis that is distinguishable by the expression of a tumor-specific antigen or a tumor-associated antigen on the cell surface thereof. Thus, in some embodiments, the present disclosure provides a method for treating cancer, the method comprising administering to a subject having or at risk of having cancer, a solid tumor and/or metastasis an effective amount of a delivery vehicle (e.g., liposomes) comprising a targeting moiety and alpha polyglutamated methotrexate (e.g., alpha PMTX disclosed herein), the cancer, solid tumor and/or metastasis being distinguishable by expression of a tumor-specific antigen or tumor-associated antigen on the cell surface thereof, and wherein the targeting moiety has specific binding affinity for an epitope on the tumor-specific antigen or tumor-associated antigen. In some embodiments, the delivery vehicle administered is a liposome. In other embodiments, the liposome is pegylated. In further embodiments, the delivery vehicle comprises a targeting moiety having specific affinity for an epitope on a cell surface antigen expressed on the surface of a cancer, solid tumor, and/or metastatic cell. In further embodiments, the targeting moiety has specific affinity for an epitope on an antigen selected from the group consisting of: GONMB, TACTD 2(TROP2), CEACAM5, EPCAM, folate receptor (e.g., folate receptor-alpha, folate receptor-beta, or folate receptor-), mucin 1(MUC-1), MUC-6, STEAP1, mesothelin, bindin 4, ENPP3, Guanylate Cyclase C (GCC), SLC44A4, NaPi2B, CD70(TNFSF7), CA9 (carbonic anhydrase), 5T4(TPBG), SLTRK6, SC-16, tissue factor, LIV-1(ZIP6), CGEN-15027, Pcadherin, fibronectin ectodomain B (ED-B), VEGFR2(CD309), tenascin, collagen IV, periostin, endothelin receptor, HER2, HER 72, EGFR, EGFRvIII, FGFR 72, FGFR3, CD ZD3, CD 36ZD 3, CD3, FGFR3, CD 36ZD 3, CD 36ZD 3, CD, CD19, CD20, CD22, CD26, CD27L, CD28, CD30, CD33, CD34, CD37, CD38, CD40, CD44, CD56, CD70, CD74, CD79, CD79b, CD98, CD105, CD133, CD138, cripto, IGF-1R, IGF-2R, IGF, EphA receptor, EphB receptor, EphA R, IGF, EphB R, IGF, EphA R, IGF, EphB R, IGF, EphUFB R, IGF, EphB R, IGF, integrin (for example, integrin. alpha. v. beta.3,. beta.5 or. alpha. v. beta.6), C antigen, Trk, VEGFR 72, PGR, VEGF-PGR, VEGF-R, IGF, PGRE, PGR, VEGF-R, IGF, PGR, and PG.
In other embodiments, the delivery vehicle is a liposome and the liposome comprises a targeting moiety having specific affinity for an epitope on a cell surface antigen selected from the group consisting of: GONMB, TACTD 2(TROP2), CEACAM5, EPCAM, folate receptor (e.g., folate receptor-alpha, folate receptor-beta, or folate receptor-), mucin 1(MUC-1), MUC-6, STEAP1, mesothelin, bindin 4, ENPP3, Guanylate Cyclase C (GCC), SLC44A4, NaPi2B, CD70(TNFSF7), CA9 (carbonic anhydrase), 5T4(TPBG), SLTRK6, SC-16, tissue factor, LIV-1(ZIP6), CGEN-15027, Pcadherin, fibronectin ectodomain B (ED-B), VEGFR2(CD309), tenascin, collagen IV, periostin, endothelin receptor, HER2, HER 72, EGFR, EGFRvIII, FGFR 72, FGFR3, CD ZD3, CD 36ZD 3, CD3, FGFR3, CD 36ZD 3, CD 36ZD 3, CD, CD19, CD20, CD22, CD26, CD27L, CD28, CD30, CD33, CD34, CD37, CD38, CD40, CD44, CD56, CD70, CD74, CD79, CD79b, CD98, CD105, CD133, CD138, cripto, IGF-1R, IGF-2R, IGF, EphA receptor, EphB receptor, EphA R, IGF, EphB R, IGF, EphA R, IGF, EphB R, IGF, EphUFB R, IGF, EphB R, IGF, integrin (for example, integrin. alpha. v. beta.3,. beta.5 or. alpha. v. beta.6), C antigen, Trk, VEGFR 72, PGR, VEGF-PGR, VEGF-R, IGF, PGRE, PGR, VEGF-R, IGF, PGR, and PG. In some embodiments, the administered delivery vehicle comprises alpha PMTX containing 4, 5, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the administered delivery vehicle comprises alpha-pentaglutaminated methotrexate. In other embodiments, the administered delivery vehicle comprises alpha-hexaglutaminated methotrexate. In some embodiments, the administered delivery vehicle comprises L α polyglutamated methotrexate. In some embodiments, the administered delivery vehicle comprises D α polyglutamated methotrexate. In some embodiments, the administered delivery vehicle comprises L and D α polyglutamated methotrexate.
In other embodiments, the present disclosure provides a method for treating cancer comprising administering to a subject having or at risk of having cancer containing cells that express a folate receptor on their cell surface an effective amount of a delivery vehicle (e.g., an antibody or liposome) comprising on its surface a targeting moiety having specific affinity for an epitope on the folate receptor and alpha polyglutamated methotrexate (e.g., alpha PMTX disclosed herein). In some embodiments, the targeting moiety is an antibody or an antigen-binding fragment of an antibody. In other embodiments, the targeting moiety has specific affinity for folate receptor alpha, folate receptor beta, or folate receptor. As disclosed herein, folate receptor targeted pegylated liposomes containing alpha polyglutamated methotrexate are capable of delivering large amounts of alpha polyglutamated methotrexate to cancer cells, and in particular cancer cells expressing folate receptors, as compared to normal cells (i.e., cells distinct from cancer cells do not actively uptake the liposomes, and/or do not express folate receptors). Any cancer that expresses a folate receptor can be treated according to the disclosed methods. It should be noted that some cancers may express the folate receptor at an early stage, while most cancers may express the folate receptor at a late stage. In some embodiments, the delivery vehicle administered is a liposome. In other embodiments, the liposome is pegylated. In some embodiments, the administered delivery vehicle comprises alpha PMTX containing 4, 5, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the administered delivery vehicle comprises alpha-pentaglutaminated methotrexate. In other embodiments, the administered delivery vehicle comprises alpha-hexaglutaminated methotrexate. In some embodiments, the administered delivery vehicle comprises L α polyglutamated methotrexate. In some embodiments, the administered delivery vehicle comprises D α polyglutamated methotrexate. In some embodiments, the administered delivery vehicle comprises L and D α polyglutamated methotrexate.
In additional embodiments, the present disclosure provides a method for cancer maintenance therapy, the method comprising administering to a subject undergoing or having undergone cancer therapy an effective amount of a liposome composition comprising liposomes containing alpha polyglutamated methotrexate (e.g., alpha PMTX disclosed herein). In some embodiments, the liposome composition administered is PLp- α PMTX, NTLp- α PMTX, NTPLp- α PMTX, TLp- α PMTX or TPLp- α PMTX. In some embodiments, the administered liposome composition comprises pegylated liposomes (e.g., PLp- α PMTX, NTPLp- α PMTX, or TPLp- α PMTX). In some embodiments, the administered liposome composition comprises a targeting moiety (e.g., TLp- α PMTX or TPLp- α PMTX) having specific affinity for an epitope on a surface antigen of the cancer cell. In some embodiments, the administered liposome composition comprises pegylated and targeted liposomes (e.g., TPLp-alpha PMTX). In some embodiments, the administered liposome composition comprises targeted liposomes and non-targeted liposomes. In some embodiments, the administered liposome composition comprises pegylated and non-pegylated liposomes. In some embodiments, the liposomes of the administered liposome composition comprise alpha polyglutamated methotrexate containing 4, 5, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the liposomes of the administered liposome composition comprise alpha-tetraglutamated methotrexate. In some embodiments, the liposomes of the administered liposome composition comprise alpha-pentaglutaminated methotrexate. In other embodiments, the liposomes of the administered liposome composition comprise alpha-hexaglutaminated methotrexate.
In some embodiments, the cancer treated by one or more of the methods disclosed herein is a solid tumor lymphoma. Examples of solid tumor lymphomas include hodgkin lymphoma, non-hodgkin lymphoma, and B-cell lymphoma.
In some embodiments, the cancer treated by one or more methods disclosed herein is bone cancer, brain cancer, breast cancer, colorectal cancer, connective tissue cancer, cancer of the digestive system, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, gastric cancer, intraepithelial tumors, melanoma neuroblastoma, non-hodgkin's lymphoma, non-small cell lung cancer, prostate cancer, retinoblastoma, or rhabdomyosarcoma.
In some embodiments, the present disclosure provides a method for treating cancer, the method comprising administering to a subject having or at risk of having cancer an effective amount of a composition comprising a delivery vehicle and alpha-polyglutamated methotrexate. In some embodiments, the administered composition comprises a pegylated delivery vehicle. In some embodiments, the administered composition comprises a targeting moiety that has a specific affinity for an epitope of an antigen on the surface of a target cell of interest (e.g., a cancer cell). In some embodiments, the delivery vehicle comprises an antibody or antigen-binding antibody fragment. In some embodiments, the composition is administered to treat a cancer selected from the group consisting of: lung cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, renal cancer, cholangiocarcinoma, gallbladder cancer, bladder cancer, sarcoma (e.g., osteosarcoma), brain cancer, central nervous system cancer, melanoma, myeloma, leukemia, and lymphoma. In some embodiments, the administered composition contains 4, 5, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the administered composition comprises alpha-tetraglutamated methotrexate. In some embodiments, the administered composition comprises alpha pentaglutamated methotrexate. In other embodiments, the administered composition comprises alpha-hexaglutaminated methotrexate.
In additional embodiments, the disclosure provides a method for treating cancer, the method comprising administering to a subject having or at risk of having cancer an effective amount of a liposome composition comprising liposomes containing alpha polyglutamated methotrexate (e.g., Lp-alpha PMTX, PLp-alpha PMTX, NTLp-alpha PMTX, NTPLp-alpha PMTX, TLp-alpha PMTX, or TPLp-alpha PMTX). In some embodiments, the liposome composition is administered to treat a cancer selected from the group consisting of: lung cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, renal cancer, cholangiocarcinoma, gallbladder cancer, bladder cancer, sarcoma (e.g., osteosarcoma), brain cancer, central nervous system cancer, melanoma, myeloma, leukemia, and lymphoma. In some embodiments, the administered liposome composition comprises pegylated liposomes (e.g., PLp- α PMTX, NTPLp- α PMTX, or TPLp- α PMTX). In some embodiments, the liposomes of the administered liposome composition comprise alpha PMTX containing 4, 5, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the liposomes of the administered liposome composition comprise alpha-tetraglutamated methotrexate. In some embodiments, the liposomes of the administered liposome composition comprise alpha-pentaglutaminated methotrexate. In other embodiments, the liposomes of the administered liposome composition comprise alpha-hexaglutaminated methotrexate.
In additional embodiments, the present disclosure provides a method for treating cancer, the method comprising administering to a subject having or at risk of having cancer an effective amount of a liposome composition comprising targeted liposomes (e.g., TLp- α PMTX or TPLp- α PMTX), wherein the liposome composition comprises liposomes comprising alpha polyglutamated methotrexate (Lp- α PMTX) and further comprising a targeting moiety having specific affinity for a surface antigen (epitope) on the cancer. In some embodiments, the liposome composition is administered to treat a cancer selected from the group consisting of: lung cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, renal cancer, cholangiocarcinoma, gallbladder cancer, bladder cancer, sarcoma (e.g., osteosarcoma), brain cancer, central nervous system cancer, melanoma, myeloma, leukemia, and lymphoma. In some embodiments, the administered liposome composition comprises pegylated liposomes (e.g., TPLp-alpha PMTX). In some embodiments, the liposomes of the administered liposome composition comprise alpha PMTX containing 4, 5, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the liposomes of the administered liposome composition comprise alpha-tetraglutamated methotrexate. In some embodiments, the liposomes of the administered liposome composition comprise alpha-pentaglutaminated methotrexate. In other embodiments, the liposomes of the administered liposome composition comprise alpha-hexaglutaminated methotrexate.
In other embodiments, the present disclosure provides a method for treating cancer, the method comprising administering to a subject having or at risk of having a cancer that expresses a folate receptor on the cell surface thereof, an effective amount of a liposome composition comprising targeted liposomes (e.g., TLp-alpha PMTX or TPLp-alpha PMTX), wherein the liposome composition comprises liposomes comprising (a) alpha polyglutamated methotrexate (alpha PMTX) and (b) a targeting moiety having specific binding affinity for a folate receptor. In some embodiments, the administered liposome composition comprises pegylated liposomes (e.g., TPLp-alpha PMTX). In some embodiments, the targeting moiety has specific binding affinity for folate receptor alpha (FR-alpha), folate receptor beta (FR-beta), and/or folate receptor (FR-). In some embodiments, the targeting moiety has specific binding affinity for folate receptor alpha (FR-alpha), folate receptor beta (FR-beta), and/or folate receptor (FR-). In some embodiments, the targeting moiety has specific binding affinity for folate receptor alpha (FR-alpha) and folate receptor beta (FR-beta). In some embodiments, the liposome composition is administered to treat a cancer selected from the group consisting of: lung cancer, pancreatic cancer, breast cancer, ovarian cancer, lung cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colon cancer, esophageal cancer, cervical cancer, renal cancer, bile duct cancer, gallbladder cancer, and hematologic malignancies. In some embodiments, the liposomes of the administered liposome composition comprise alpha PMTX containing 4, 5, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the liposomes of the administered liposome composition comprise alpha-tetraglutamated methotrexate. In some embodiments, the liposomes of the administered liposome composition comprise alpha-pentaglutaminated methotrexate. In other embodiments, the liposomes of the administered liposome composition comprise alpha-hexaglutaminated methotrexate.
In some embodiments, the present disclosure provides a method for treating an immune system disorder (e.g., an autoimmune disease, such as rheumatoid arthritis), the method comprising administering to a subject having or at risk of having an immune system disorder an effective amount of a delivery vehicle (e.g., an antibody or liposome) comprising alpha polyglutamated methotrexate (e.g., an alpha PMTX disclosed herein). In some embodiments, the delivery vehicle is an antibody (e.g., a full-length IgG antibody, a bispecific antibody, or an scFv). In some embodiments, the delivery vehicle is a liposome (e.g., Lp- α PMTX, such as PLp- α PMTX, NTLp- α PMTX, NTPLp- α PMTX, TLp- α PMTX, or TPLp- α PMTX). In some embodiments, the administered delivery vehicle is pegylated. In some embodiments, the administered delivery vehicle is non-pegylated. In further embodiments, the administered delivery vehicle comprises a targeting moiety having specific affinity for an epitope of an antigen on the surface of an immune cell associated with an immune system disorder. In some embodiments, the targeting moiety is an antibody or antigen binding antibody fragment. In some embodiments, the administered delivery vehicle comprises alpha PMTX containing 4, 5, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the administered delivery vehicle comprises alpha-pentaglutaminated methotrexate. In other embodiments, the administered delivery vehicle comprises alpha-hexaglutaminated methotrexate. In some embodiments, the administered delivery vehicle comprises L α polyglutamated methotrexate. In some embodiments, the administered delivery vehicle comprises D α polyglutamated methotrexate. In some embodiments, the administered delivery vehicle comprises L and D α polyglutamated methotrexate. In some embodiments, the autoimmune disease is rheumatoid arthritis.
In some embodiments, the present disclosure provides a method for treating an infectious disease (e.g., HIV), the method comprising administering to a subject having or at risk of having an infectious disease an effective amount of a delivery vehicle (e.g., an antibody or liposome) comprising alpha polyglutamated methotrexate (e.g., alpha PMTX disclosed herein). In some embodiments, the delivery vehicle is an antibody (e.g., a full-length IgG antibody, a bispecific antibody, or an scFv). In some embodiments, the delivery vehicle is a liposome (e.g., Lp- α PMTX, such as PLp- α PMTX, NTLp- α PMTX, NTPLp- α PMTX, TLp- α PMTX, or TPLp- α PMTX). In some embodiments, the administered delivery vehicle is pegylated. In some embodiments, the administered delivery vehicle is non-pegylated. In further embodiments, the administered delivery vehicle comprises a targeting moiety having specific affinity for an epitope of an antigen on the surface of a pathogen associated with an infectious disease. In some embodiments, the targeting moiety is an antibody or antigen binding antibody fragment. In some embodiments, the administered delivery vehicle comprises alpha PMTX containing 4, 5, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the administered delivery vehicle comprises alpha-pentaglutaminated methotrexate. In other embodiments, the administered delivery vehicle comprises alpha-hexaglutaminated methotrexate. In some embodiments, the administered delivery vehicle comprises L α polyglutamated methotrexate. In some embodiments, the administered delivery vehicle comprises D α polyglutamated methotrexate. In some embodiments, the administered delivery vehicle comprises L and D α polyglutamated methotrexate.
In some embodiments, the delivery vehicle administered is a liposome. In other embodiments, the liposome is pegylated. In further embodiments, the delivery vehicle comprises on its surface a targeting moiety having specific affinity for an epitope on the surface of a target cell of interest. In other embodiments, the delivery vehicle comprises a targeting moiety having specific affinity for an epitope on a cell surface antigen selected from the group consisting of: GONMB, TACTD 2(TROP2), CEACAM5, EPCAM, folate receptor (e.g., folate receptor-alpha, folate receptor-beta, or folate receptor-), mucin 1(MUC-1), MUC-6, STEAP1, mesothelin, bindin 4, ENPP3, Guanylate Cyclase C (GCC), SLC44A4, NaPi2B, CD70(TNFSF7), CA9 (carbonic anhydrase), 5T4(TPBG), SLTRK6, SC-16, tissue factor, LIV-1(ZIP6), CGEN-15027, Pcadherin, fibronectin ectodomain B (ED-B), VEGFR2(CD309), tenascin, collagen IV, periostin, endothelin receptor, HER2, HER 72, EGFR, EGFRvIII, FGFR 72, 3, FGFR3, CD 36ZD 3, CD 36ZD 3, CD 36, CD19, CD20, CD22, CD26, CD27L, CD28, CD30, CD33, CD34, CD37, CD38, CD40, CD44, CD56, CD70, CD74, CD79, CD79b, CD98, CD105, CD133, CD138, cripto, IGF-1R, IGF-2R, IGF, EphA receptor, EphB receptor, EphA R, IGF, EphB R, IGF, EphA R, IGF, EphB R, IGF, EphUFB R, IGF, EphB R, IGF, integrin (for example, integrin. alpha. v. beta.3,. beta.5 or. alpha. v. beta.6), C antigen, Trk, VEGFR 72, PGR, VEGF-PGR, VEGF-R, IGF, PGRE, PGR, VEGF-R, IGF, PGR, and PG.
In other embodiments, the delivery vehicle is a liposome and the liposome comprises a targeting moiety having specific affinity for an epitope on a cell surface antigen selected from the group consisting of: GONMB, TACTD 2(TROP2), CEACAM5, EPCAM, folate receptor (e.g., folate receptor-alpha, folate receptor-beta, or folate receptor-), mucin 1(MUC-1), MUC-6, STEAP1, mesothelin, bindin 4, ENPP3, Guanylate Cyclase C (GCC), SLC44A4, NaPi2B, CD70(TNFSF7), CA9 (carbonic anhydrase), 5T4(TPBG), SLTRK6, SC-16, tissue factor, LIV-1(ZIP6), CGEN-15027, Pcadherin, fibronectin ectodomain B (ED-B), VEGFR2(CD309), tenascin, collagen IV, periostin, endothelin receptor, HER2, HER 72, EGFR, EGFRvIII, FGFR 72, 3, FGFR3, CD 36ZD 3, CD 36ZD 3, CD 36, CD19, CD20, CD22, CD26, CD27L, CD28, CD30, CD33, CD34, CD37, CD38, CD40, CD44, CD56, CD70, CD74, CD79, CD79b, CD98, CD105, CD133, CD138, cripto, IGF-1R, IGF-2R, IGF, EphA receptor, EphB receptor, EphA R, IGF, EphB R, IGF, EphA R, IGF, EphB R, IGF, EphUFB R, IGF, EphB R, IGF, integrin (for example, integrin. alpha. v. beta.3,. beta.5 or. alpha. v. beta.6), C antigen, Trk, VEGFR 72, PGR, VEGF-PGR, VEGF-R, IGF, PGRE, PGR, VEGF-R, IGF, PGR, and PG.
In some embodiments, the present disclosure provides for the use of a composition comprising alpha polyglutamated methotrexate for the manufacture of a medicament for the treatment of a hyperproliferative disease. In some embodiments, the alpha polyglutamated methotrexate comprises 5 or more glutamyl groups. In some embodiments, the alpha polyglutamated methotrexate is pentaglutamated or hexaglutamated. In some embodiments, the alpha-polyglutamated methotrexate is polyglutamated Methotrexate (MTX), Methotrexate (MTX). In some embodiments, the alpha-polyglutamated methotrexate is in a liposome. In some embodiments, the hyperproliferative disease is cancer. In some embodiments, the cancer is selected from the group consisting of: lung cancer (e.g., non-small cell lung cancer), pancreatic cancer, breast cancer, ovarian cancer, lung cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colon cancer, esophageal cancer, cervical cancer, renal cancer, bile duct cancer, gallbladder cancer, and hematologic malignancies. In some embodiments, the cancer is pancreatic cancer. In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is pancreatic cancer. In some embodiments, the cancer is triple negative breast cancer. In some embodiments, the cancer is lung cancer. In some embodiments, the cancer is non-small cell lung cancer. In some embodiments, the cancer is leukemia or lymphoma. In some embodiments, the hyperproliferative disease is an autoimmune disease. In some embodiments, the hyperproliferative disease is rheumatoid arthritis.
The disclosed methods can be practiced in any subject that may benefit from the delivery of compositions contemplated herein (e.g., alpha polyglutamated methotrexate compositions, such as liposomes containing pentaglutamated or hexaglutamated MTX). Mammalian subjects, and particularly human subjects, are preferred. In some embodiments, subjects also include animals, such as domestic pets (e.g., dogs, cats, rabbits, and ferrets), livestock or domestic animals (e.g., cattle, pigs, sheep, chickens, and other poultry), horses (e.g., thoroughbred horses), laboratory animals (e.g., mice, rats, and rabbits), and other mammals. In other embodiments, the subject includes fish and other aquatic species.
The subject to which the agent is delivered may be a normal subject. Alternatively, the subject may have or be at risk of developing a condition that can be diagnosed or that can benefit from delivery of one or more compositions provided. In some embodiments, such disorders include cancer (e.g., solid tumor cancer or non-solid cancer, such as leukemia). In some embodiments, these conditions (e.g., cancer) involve cells expressing an antigen that can be specifically bound by the targeted pegylated liposomal alpha-polyglutamated methotrexate disclosed herein. In other embodiments, these antigens specifically bind to targeted pegylated liposomal alpha-polyglutamated methotrexate and internalize the methotrexate into the cell. In some embodiments, the targeted pegylated liposomal alpha-polyglutamated methotrexate specifically binds to folate receptors expressed on the surface of cancer cells (e.g., folate receptor alpha (FR-alpha), folate receptor beta (FR-beta), and folate receptor (FR-)).
Tests for diagnosing conditions treatable with the provided compositions are known in the art and familiar to medical practitioners. Commercially available antibodies can be used to determine whether a cell type expresses a folate receptor. These laboratory tests include, but are not limited to, microscopic analysis, culture-dependent tests (e.g., culture), and nucleic acid detection tests. These include wet scaffolds, dye enhanced microscopy, immuno microscopy (e.g., FISH), hybridization microscopy, particle agglutination, enzyme-linked immunosorbent assay, urine screening tests, DNA probe hybridization, and serological tests. In addition to performing the above laboratory tests, medical practitioners typically record a complete medical history and perform a comprehensive physical examination.
A subject having cancer can be, for example, a subject having detectable cancer cells. For example, a subject at risk for cancer may be a subject with a higher than normal probability of developing cancer. Such subjects include, for example, subjects with genetic abnormalities that have been shown to be more likely to be associated with developing cancer, subjects with familial predisposition to cancer, subjects exposed to carcinogens (i.e., carcinogens) such as tobacco, asbestos, or other chemical toxins, and subjects who have previously been treated for cancer and significantly remitted.
In some embodiments, the present disclosure provides methods for selectively delivering folate receptor targeted pegylated liposomal alpha-polyglutamated methotrexate to tumor cells that express folate receptors on their surface at a higher rate (e.g., at least two times higher, at least three times higher, at least four times higher, or at least five times higher) than cells that do not express folate receptors on their cell surface. In some embodiments, the pegylated liposomes delivered comprise alpha polyglutamated MTX. In some embodiments, the delivered pegylated liposomes comprise L-alpha polyglutamated MTX. In some embodiments, the delivered pegylated liposomes comprise D-alpha polyglutamated MTX.
i. Combination therapy
In certain embodiments, the method or treatment comprises administering at least one additional therapeutic agent in addition to administering the alpha polyglutamated MTX compositions described herein. The additional therapeutic agent may be administered prior to, concurrently with, and/or after administration of the alpha polyglutamated MTX composition. The additional therapeutic agent may be associated with the alpha polyglutamated MTX delivery vehicle present in a solution containing the alpha polyglutamated MTX delivery vehicle (e.g., co-encapsulated with the alpha polyglutamated MTX in a liposome), or in a separate formulation from the composition containing the alpha polyglutamated MTX composition. Also provided are pharmaceutical compositions comprising the polypeptide or agent and one or more additional therapeutic agents. In some embodiments, the at least one additional therapeutic agent comprises 1, 2, 3, or more additional therapeutic agents.
Combination therapies using two or more therapeutic agents often use agents that act through different mechanisms of action, although this is not necessary. Combination therapy with agents having different mechanisms of action may produce additive or synergistic effects. Combination therapy may allow the use of lower doses of each dose than those used in monotherapy, thereby reducing toxic side effects and/or increasing the therapeutic index of one or more polypeptides or agents. Combination therapy can reduce the likelihood that resistant cancer cells will develop. In some embodiments, the combination therapy includes a therapeutic agent that affects an immune response (e.g., enhances or activates a response) and a therapeutic agent that affects (e.g., inhibits or kills) a tumor/cancer cell.
In some embodiments, the present disclosure provides a method for treating cancer comprising administering an effective amount of an alpha polyglutamated methotrexate composition and a biologic disclosed herein. In some embodiments, the alpha-polyglutamated methotrexate is administered in combination with a therapeutic antibody. In other embodiments, the alpha polyglutamated methotrexate is administered in combination with an anti-CD antibody (e.g., rituximab) or an antibody that binds an immune checkpoint protein (e.g., CTLA4, PD1, PDL1, and TIM 3). In other embodiments, the alpha-polyglutamated methotrexate is administered in combination with an fc fusion protein (e.g., etanercept).
In some embodiments, the present disclosure provides a method for treating an immune system disorder, the method comprising administering an effective amount of an alpha polyglutamated methotrexate composition and a biologic agent disclosed herein. In some embodiments, the alpha-polyglutamated methotrexate is administered in combination with a therapeutic antibody. In other embodiments, the alpha-polyglutamated methotrexate is administered in combination with an anti-TNF antibody (e.g., adalimumab). In some embodiments, the alpha-polyglutamated methotrexate is administered in combination with a fc fusion protein (e.g., etanercept).
In some embodiments of the methods described herein, the combination of an alpha PMTX composition described herein and at least one additional therapeutic agent produces an additive or synergistic result. In some embodiments, the combination therapy results in an increase in the therapeutic index of the alpha PMTX or agent. In some embodiments, the combination therapy results in an increase in the therapeutic index of the one or more additional therapeutic agents. In some embodiments, the combination therapy results in reduced toxicity and/or side effects of the alpha PMTX or agent. In some embodiments, the combination therapy results in a reduction in toxicity and/or side effects of one or more additional therapeutic agents.
In some embodiments, in addition to administering the α polyglutamated MTX compositions described herein, the methods or treatments described herein further comprise administering at least one additional therapeutic agent selected from the group consisting of: anti-tubulin agents, auristatins, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents (e.g., platinum complexes such as cisplatin, mono (platinum), bis (platinum), and trinuclear platinum complexes and carboplatin), anthracyclines, antibiotics, antifolates (e.g., polyglutamated or non-polyglutamated antifolates), antimitotics (e.g., vinca alkaloids such as vincristine, vinblastine, vinorelbine, or vindesine), radiosensitizers, steroids, taxanes, topoisomerase inhibitors (e.g., doxorubicin HCl, daunorubicin citrate, mitoxantrone HCl, actinomycin D, etoposide, topotecan HCl, teniposide (VM-26), and irinotecan), antimetabolites, chemotherapeutic sensitizers, duocarmycin, etoposide, fluorinated pyrimidines, ionophores, pharmaceutical compositions, and methods of the use thereof, Levofloxacin, nitrosoureas, platinum, purine antimetabolites, PARP inhibitors, and puromycin. In certain embodiments, the second therapeutic agent is an alkylating agent, an antimetabolite, an antimitotic agent, a topoisomerase inhibitor, or an angiogenesis inhibitor.
Therapeutic agents that can be administered in combination with the alpha PMTX compositions described herein include chemotherapeutic agents. Thus, in some embodiments, the methods or treatments described herein further comprise administering an alpha PMTX composition described herein in combination with or in combination with a chemotherapeutic agent. Treatment with the alpha PMTX composition can occur before, concurrently with, or after administration of chemotherapy. Combined administration may include co-administration (whether in a single pharmaceutical formulation or using separate formulations), or sequential administration in any order, but is typically used over a period of time such that all active agents are capable of exerting their biological activities simultaneously. The preparation and schedule of administration of such chemotherapeutic agents may be used according to manufacturer's instructions or determined empirically by a skilled practitioner. Preparation and dosing schedules for this Chemotherapy are also described in The Chemotherapy Source Book, 4 th edition, 2008, m.c. perry, editions, Lippincott, Williams & Wilkins, philiadelphia, PA.
Chemotherapeutic agents useful in the present invention include, but are not limited to, alkylating agents, such as thiotepa and Cyclophosphamide (CYTOXAN); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridine hospitals such as benzodopa (benzodopa), carboquone (carboquone), midodopa (meteredopa), and ulidopa (uredopa); ethyleneimine and methylmelamines (melamelamines), including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine; nitrogen mustards, such as chlorambucil, chlorambucil (chlorenaphazine), chlorophosphamide, estramustine (estramustine), isocyclophosphamine, dichloromethyldiethanamine (mechlorethamine), mechlorethamine hydrochloride, melphalan, neoenticine (novembichin), benzene mustard cholesterol (phereneine), prednimustine (prednimustine), trofosfamide (trofosfamide), uracil mustard; nitrosoureas such as carmustine, chlorozotocin (chlorozotocin), fotemustine (fotemustine), lomustine, nimustine (nimustine), ramustine (ranimustine); antibiotics, such as aclacinomycins (aclacinomycins), actinomycins, antromycin (aurramycin), azaserine, bleomycin (bleomycin), actinomycin (cactinomycin), calicheamicin (calicheamicin), karabinin (carahemin), carminomycin (caminomycin), carcinomycin (carzinophilin), chromomycin (chromomycins), dactinomycin, daunomycin (detritucin), ditorelbirubicin (detubicin), 6-diazo-5-oxo-L-norleucine, adriamycin, epirubicin, esorubicin (esorubicin), idarubicin (idarubicin), marijudamycin (marijubamycin), mitomycin, mycophenolic acid, nogaxomycin (nogalamoutamycin), olivomycin (olivomycin), plemycin (polypicins), puromycin (poromycin), pleomycin), streptomycin (streptomycin), streptomycin (streptomycin, streptomyc, Neat stastatin (zinostatin), zorubicin (zorubicin); antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues, such as denopterin, methotrexate, pteropterin, tripropterin Metrizasa (trimetrexate); purine analogues, such as fludarabine (fludarabine), 6-mercaptopurine, thioprimine (thiaapine), thioguanine (thioguanine); pyrimidine analogs such as ancitabine (ancitabine), azacitidine, 6-azauridine, carmofur (carmofur), cytarabine, dideoxyuridine (dideoxyuridine), doxifluridine, enocitabine (enocitabine), floxuridine, 5-FU; androgens, such as carroterone (calusterone), dromostanolone propionate (dromostanolone), epithioandrostanol (epitiostanol), mepiquitane (mepitistane), testolactone (testolactone); anti-adrenergic agents, such as aminoglutethimide, mitotane, trilostane; folic acid supplements, such as folinic acid (frilic acid); acetoglucurolactone (acegultone); an aldehydic phosphoramide glycoside; (ii) aminolevulinic acid; ambridine (amsacrine); betributil (betlabucil); bisantrene; edatrexate (edatraxate); defluoromine (defofamine); colchicine (de mecolcine); diazaquinone (diaziqutone); eflornithine (elformithine); ammonium etitanium acetate; etoglut (etoglucid); gallium nitrate; a hydroxyurea; lentinan; lonidamine (lonidamine); mitoguazone (mitoguzone); mitoxantrone (mitoxantrone); mopidamol (mopidamol); diamine nitracridine (nitrarine); pentostatin (pentostatin); methionine mustard (phenamett); pirarubicin (pirarubicin); podophyllinic acid; 2-ethyl linear hydrazine; procarbazine; PSK; razoxane (rizoxane); azofurans (sizofurans); germanium spiroamines (spirogyranium); tenuazonic acid (tenuazonic acid); triimine quinone (triaziquone); 2,2' -trichlorotriethylamine; urethane (urethan); vindesine (vindesine); dacarbazine (dacarbazine); mannomustine (mannomustine); dibromomannitol (mitobronitol); dibromodulcitol (mitolactol); pipobromane (pipobroman); gatifloxacin (gacytosine); cytarabine (Ara-C); taxanes, e.g. paclitaxel
Figure BDA0002715283060000921
And docetaxel
Figure BDA0002715283060000922
Chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; platinum analogs, such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; novier; norfloxacin (novantrone); (ii) teniposide; daunomycin; methotrexate; ibandronate (ibandronate); CPT 11; topoisomerase inhibitor RFS 2000; difluoromethyl ornithine (DMFO); retinoic acid; esperamicins (esperamicins); capecitabine (XELODA); anti-hormonal agents such as tamoxifen, raloxifene, aromatase inhibiting 4(5) -imidazole, 4-hydroxyttamoxifen, trioxifene, raloxifene (keoxifene), LY117018, onapristone, and toremifene (FARESTON); antiandrogens, such as flutamide (flutamide), nilutamide (nilutamide), bicalutamide (bicalutamide), leuprolide acetate and goserelin (goserelin); and a pharmaceutically acceptable salt, acid or derivative of any of the foregoing. In certain embodiments, the additional therapeutic agent is cisplatin. In certain embodiments, the additional therapeutic agent is carboplatin. In other embodiments, the additional therapeutic agent is oxaliplatin (oxaloplatin).
V. kit comprising an alpha PMTX composition
The present disclosure also provides kits comprising the alpha PMTX compositions described herein and useful for performing the methods described herein. In certain embodiments, the kit comprises at least one purified alpha PMTX composition in one or more containers.
In some embodiments, the kit comprises a dose (e.g., for treatment or diagnosis) of at least one alpha PMTX composition (e.g., an alpha PMTX liposome) or pharmaceutical formulation thereof as disclosed herein. The kit may also include suitable packaging and/or instructions for using the composition. The kit may further comprise a device for delivering the composition or pharmaceutical formulation thereof, such as an injection syringe or other device as described herein and known to those skilled in the art. One skilled in the art will readily recognize that the disclosed alpha PMTX compositions can be readily incorporated into one of the established kit forms well known in the art.
Kits comprising the alpha PMTX compositions and at least one additional therapeutic agent are also provided. In certain embodiments, the second (or more) therapeutic agent is an antimetabolite. In certain embodiments, the second (or more) therapeutic agent is a chemotherapeutic agent.
The following examples are intended to illustrate, but not to limit the disclosure in any way, state, or form, either explicitly or implicitly. While they are exemplary of the embodiments that may be used, other procedures, methods, or techniques known to those of skill in the art may alternatively be used without departing from the scope of the present disclosure.
Fig. 1B-1N show the chemical formulas of exemplary alpha polyglutamates contemplated by the present disclosure.
Examples
Example 1: liposome gamma polyglutamated pemetrexed composition
The method comprises the following steps:
production of gamma-hexaglutaminated pemetrexed (gamma HgPTX) liposomes
Briefly, gamma-hexaglutaminated pemetrexed (gGM6) and D-alpha-hexaglutaminated pemetrexed (gDGM6) were encapsulated in liposomes by the following procedure. First, the lipid components of the liposome membrane were weighed out and combined as a concentrated ethanol solution at a temperature of about 65 ℃. In this example, the lipids used were hydrogenated soy phosphatidylcholine, cholesterol and DSPE-PEG-2000(1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [ methoxy (polyethylene glycol) -2000 ]). The molar ratio of HSPC to cholesterol to PEG-DSPE is about 3:2: 0.15. Next, gGM6 or gDGM6 were dissolved in 5% dextrose at a concentration of 100-150mg/ml, pH 6.5-6.9. The drug solution was heated to 65 ℃. The ethanolic lipid solution was injected into gGM6 or gDGM6 solution using a small bore needle. During this step, the drug solution was thoroughly stirred using a magnetic stirrer. Mixing is performed at elevated temperatures (63-72 ℃) to ensure that the lipids are in a liquid crystalline state (as opposed to a gel state achieved at temperatures below the lipid transition temperature Tm-51-54 ℃). As a result, the lipids hydrate and form multiple bilayer (multilamellar) vesicles (MLVs) containing gGM6 or gDGM6 in the aqueous core.
Shrinking MLV using filter extrusion
MLVs are fragmented into monolayer (single bilayer) vesicles of the desired size by high pressure extrusion using three passes through a stack of (track etched polycarbonate) membranes. The first pass was through a stacked membrane consisting of two layers with a pore size of 200 nm. The other two passes were made through a stacked membrane consisting of three layers with a pore size of 100 nm. During extrusion, the temperature is kept above Tm to ensure plasticity of the lipid film. Due to extrusion, large MLVs that are not uniform in size and layer become small, uniform (90-125nm) unilamellar vesicles (ULVs), which sequester the drug inside. Hydrodynamic dimensions (diameter) at 25 ℃ were measured in a quartz microcuvette using a Malvern Zetasizer Nano ZS instrument (Southborough, MA) with a backscatter detector (90 °). Samples were diluted 50-fold in the formulation matrix prior to analysis.
Purification of liposomes
After generating ULVs containing gGM6 or gDGM6, the liposome-free drug was removed using tangential flow diafiltration against a suitable buffer for small volumes of column or for large volumes. Although any buffer solution may be used, in this example, the buffer used is 5mM HEPES, 145mM sodium chloride (pH 6.7). After completion of purification, filter sterilization was performed using a 0.22 micron filter.
Production of alpha-hexaglutaminated pemetrexed (alpha HgPTX) liposomes
Briefly, L α hexaglutaminated pemetrexed (aG6) and D α hexaglutaminated pemetrexed (aDG6) were encapsulated in liposomes by the following procedure. First, the lipid components of the liposome membrane were weighed out and combined as a concentrated ethanol solution at a temperature of about 65 ℃. In this example, the lipids used were hydrogenated soy phosphatidylcholine, cholesterol and DSPE-PEG-2000(1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [ methoxy (polyethylene glycol) -2000 ]). The molar ratio of HSPC to cholesterol to PEG-DSPE is about 3:2: 0.15. Next, aG6 or aDG6 was dissolved in 5% dextrose at a concentration of 150mg/ml, pH 6.5-6.9. The drug solution was heated to 65 ℃. The ethanol lipid solution was injected into the aG6 or aDG6 solution using a small bore needle. During this step, the drug solution was thoroughly stirred using a magnetic stirrer. Mixing is performed at elevated temperatures (63-72 ℃) to ensure that the lipids are in a liquid crystalline state (as opposed to a gel state achieved at temperatures below the lipid transition temperature Tm-51-54 ℃). As a result, the lipids hydrate and form multiple bilayer (multilamellar) vesicles (MLVs) containing aG6 or aDG6 in the aqueous core.
Shrinking MLV using filter extrusion
MLVs are fragmented into monolayer (single bilayer) vesicles of the desired size by high pressure extrusion using three passes through a stack of (track etched polycarbonate) membranes. The first pass was through a stacked membrane consisting of two layers with a pore size of 200 nm. The other two passes were made through a stacked membrane consisting of three layers with a pore size of 100 nm. During extrusion, the temperature is kept above Tm to ensure plasticity of the lipid film. Due to extrusion, large MLVs that are not uniform in size and layer become small, uniform (90-125nm) unilamellar vesicles (ULVs), which sequester the drug inside. Hydrodynamic dimensions (diameter) at 25 ℃ were measured in a quartz microcuvette using a Malvern Zetasizer Nano ZS instrument (Southborough, MA) with a backscatter detector (90 °). Samples were diluted 50-fold in the formulation matrix prior to analysis.
Purification of liposomes
After generation of ULVs containing aG6 or aDG6, the extra-liposomal gG6 was removed using tangential flow diafiltration against a suitable buffer for small volumes of column or for large volumes. Although any buffer solution may be used, in this example, the buffer used is 5mM HEPES, 145mM sodium chloride (pH 6.7). After completion of purification, filter sterilization was performed using a 0.22 micron filter. Typical characteristics of the liposome derivatives are shown in the table below.
Figure BDA0002715283060000941
Dose response study of alpha HGP (hexaglutaminated pemetrexed) and liposomes
Passage on day 3 (48 hours) and day 4 (72 hours)
Figure BDA0002715283060000942
(CTG) luminescence cell viability assay to determine cell viability. This assay determines the number of viable cells in culture based on quantifying the ATP present therein, which in turn indicates the presence of metabolically active cells. CTG assays use luciferase as readout. To assess cell viability, use was made of
Figure BDA0002715283060000943
Luminocyte viability assay study dose-response inhibition of pemetrexed, HGP and liposomes on growth of different cancer cells. Human cancer cells were harvested, counted and seeded at the same cell density on day 0. On day 1, a series of 8 dilutions of each test article was added to the cells. Dose response curves were generated and fitted using GraphPad Prism and the IC50 for each test article was calculated. The lower the IC50, the more effective the test article is in inhibiting cancer cell growth.
On day 0, cells were plated at 5x 10 per well4The cell density of each cell was seeded into 100. mu.l of fresh medium in a 96-well plate. 8 serial 2-fold dilutions of each test article in culture medium were generated and added to the cells in triplicate on day 1. In addition, cells from three wells were treated with vehicle only (HBS for free drug or empty liposomes for liposomal HGP) as control.
On day 3 and 4, 100. mu.l of each well was added
Figure BDA0002715283060000944
Reagents and incubated for 15 minutes at room temperature. Luciferase luminescence was recorded for each well. In addition, 8 serial 2-fold dilutions of vehicle (HBS or empty liposomes) in culture medium were added to the empty wells and included in the assay to generate background luminescence signal. Luciferase signals were normalized by subtracting the background luminescence signal from the readings, respectively.
Human normal primary bone marrow CD34+ cells were obtained from ATCC (ATCC accession No. PCS-800-012). Cells were thawed at 37 ℃ for 1 minute and then placed on ice. The cells were then resuspended in StemBan SFEM (Stem cell technology catalog number 9650) plus 10% heat-inactivated fetal bovine serum (Corning 35-015-CV). Cells were plated at 2.5X104The density of individual cells/well was seeded into 96-well culture plates. The next day, viable cells were collected via centrifugation and resuspended at a density of 2.5 × 104 cells/well in neutrophil growth medium (StemBan SFEM plus 10% heat-inactivated fetal bovine serum plus 100ng/ml human stem cell factor (Sigma Cat. No. H8416), 20ng/ml human granulocyte colony stimulating factor (Sigma Cat. No. H5541), and 10ng/ml human recombinant IL3(Sigma SRP 3090). The cells were incubated at 37 ℃ for 10 days 4The density of individual cells/well was seeded in 96-well plates and incubated overnight at 37 ℃. The following day, the test article or vehicle was resuspended in neutrophil growth medium and added to the plate. The cells were then incubated at 37 ℃ for 48 hours or 72 hours, and then assayed at each time point using the Cell Titer Glo assay (Promega catalog No. G7572).
The methods used for the cell lines AML12 (non-cancerous hepatocytes) and CCD841 (non-cancerous colonic epithelial cells) are similar to those used for cancer cells.
Results
In a set of dose-response experiments, 6 cell lines representing different types of cancer were studied, namely HT-29 (colon cancer), H2342(NSCLC, adenocarcinoma subtype), H292(NSCLC, adenocarcinoma subtype), SW620(CRC), H1806 (triple negative breast cancer) and OAW28 (ovarian cancer) (fig. 2). The treatment consisted of 48 hours exposure using 2 different encapsulated derivatives of liposomal α pemetrexed hexaglutamate, namely liposomal α L hexaglutamate (liposomal aG6) and its mirror image liposomal α D hexaglutamate (liposomal aDG6), also known as its corresponding enantiomer.
The relative potency of the above derivatives compared to pemetrexed within 48 hours after exposure is shown in figure 2. For each cell line, as shown in this figure, the relative potency of treatment with various derivatives was calculated by dividing IC50 for pemetrexed by IC50 for liposomal alpha pemetrexed hexaglutamate. As shown in this figure, the potency of liposomal alpha pemetrexed hexaglutamate far exceeded that of pemetrexed in all cell lines. For example, consider NSCLC cell line H292. As shown, the efficacy of liposomal alpha pemetrexed hexaglutamate was greater than or equal to 50 times that of pemetrexed. This indicates that 2% or lower doses of liposomal alpha pemetrexed hexaglutamate may have the same therapeutic effect as 100% doses of pemetrexed.
As described in some examples, increased payload uptake can be achieved by targeting the liposomal delivery vehicle with an antibody, such as folate receptor alpha. By way of example, in the next two experiments, liposomes L γ G6/Lps Hexa gG6 were encapsulated using the method described previously above. Subsequently, as shown in fig. 3 and 4, respectively, pemetrexed, liposomal γ pemetrexed Hexa glutamate derivative (liposomal L γ G6/Lps Hexa gG6), and folate receptor α -targeting liposomal L γ G6 (liposomal gG6-FR1Ab), free (unencapsulated) L γ G6 were tested for cytotoxic activity against representative cell lines among non-small cell lung cancer cells (NCI-H2342) and colorectal cancer cells (HT-29). These data indicate that liposomal L γ pemetrexed hexaglutamate and folate receptor α targeting liposomal L γ pemetrexed hexaglutamate are more effective than pemetrexed in both cell lines. In general, folate receptor alpha antibody targeted liposomes showed the highest potency. In contrast, free L γ G6 has the lowest potency due to its inability to transport efficiently across cell membranes.
Cancer cell viability studies comparing the cytotoxic activity of liposomal alpha pemetrexed hexaglutamate derivatives (liposomal L α G6/Lps Hexa aG6 and liposomal D α G6/Lps Hexa agg 6) and pemetrexed on representative cell lines in breast, lung and ovarian cancers are shown in fig. 5-7. These data indicate that both liposomal α L pemetrexed hexaglutamate and liposomal α D pemetrexed hexaglutamate are more effective than pemetrexed. Furthermore, as an indicator of efficacy, experimental results for the same cell line at different dose levels in the range of 16 to 128nM are depicted in fig. 8-10. As shown in the figures, liposomal α L pemetrexed hexaglutamate and liposomal α D pemetrexed hexaglutamate are superior to pemetrexed in inhibiting cancer cells for lung and breast cancer cell lines in each of these dose ranges. In ovarian cancer cell lines, pemetrexed at 128nM dose appeared to be as effective as liposomal α pemetrexed hexaglutamate, while liposomal α pemetrexed hexaglutamate at 32nM and 64nM doses had better therapeutic efficacy than pemetrexed; at 16nM, the therapeutic effect was lower and similar in magnitude for liposomal alpha pemetrexed hexaglutamate and pemetrexed.
The major toxicity observed in patients treated with pemetrexed is myelosuppression, which is manifested as a decrease in blood counts, including neutrophil counts (a type of white blood cell). There are also some adverse effects on the intima of the mouth and intestines, which are manifested as diarrhea and mucositis, and in some cases, adverse effects on the liver. To assess the above toxicity, the treatment of liposomal α pemetrexed hexaglutamate derivatives (L and D) and pemetrexed was measured at 48 hours on CD34+ cells differentiated into neutrophils, CCD841 colonic epithelial cells, and AML12 hepatocytes. As shown in fig. 11, liposomal alpha pemetrexed hexaglutamate is significantly less toxic to differentiated human neutrophils compared to pemetrexed. This is also supported by the better retained neutrophil counts after treatment with liposomal α L pemetrexed hexaglutamate or liposomal α D pemetrexed hexaglutamate at doses ranging from 16nM to 128nM compared to pemetrexed (fig. 12). Strikingly, there did not appear to be any toxicity to hepatocytes after treatment with liposomal L α pemetrexed hexaglutamate or liposomal α D pemetrexed hexaglutamate at the dose levels studied (fig. 13). In contrast, pemetrexed at all doses studied resulted in approximately 40% reduction in hepatocyte counts. Finally, the same trend was observed after treatment of epithelial colon cells (fig. 14). As shown in this figure, all doses of pemetrexed studied resulted in a reduction in cell number of approximately ≧ 50% as compared to a reduction of approximately 20% or less after treatment with liposomal α L pemetrexed hexaglutamate and liposomal α D pemetrexed hexaglutamate.
Example 2: polyglutamated antifolate-cisplatin complex (PGPD)
The method comprises the following steps:
folic acid analogs (also known as antifolates) have been an important anticancer therapy for the past 70 years. Such anticancer drugs used in this case interfere with various enzymes in important folate metabolic pathways. This can lead to impaired pyrimidine and purine (DNA and RNA) synthesis, impaired glycine and serine metabolism, impaired redox reactions and impaired intracellular methylation processes.
In clinical practice, antifolates (such as pemetrexed and methotrexate) are commonly used in combination with platinum agents (such as cisplatin and carboplatin). The combination results in enhanced efficacy. In this case, we set out to co-encapsulate polyglutamate with platinum agents in specific ratios to facilitate controlled delivery of a predetermined ratio of two anticancer drugs, a polyglutamated antifolate and a platinum analog. It has been surprisingly found that long forms of polyglutamated antifolates (e.g., pentaglutamated antifolates) form complexes with cisplatin that are stable at high pH, and that such complexes dissociate into polyglutamate and cisplatin at low pH. Low pH is thought to occur in many tumor cells and tumor cell environments, particularly in hypoxic environments. The use of this discovery provides the ability to facilitate delivery of a combination of alpha polyglutamated pemetrexed (alpha PPMX) and a therapeutic agent, such as cisplatin, to a target cell, such as a tumor cell, and release the drug from the complex under physiologically relevant low pH conditions.
Production of polyglutamated antifolate-DDAP (cisplatin) Complex (PGPD)
To generate (polyglutamated antifolate-cisplatin DDAP complex), alpha-hexaglutamate (aG6) and diaminedicarboxylic acid platinum (DDAP) were used. The complexing process depends on the presence of the platinum chloride compound and the pH conditions. Complexation is achieved by nucleophilic attack of one or both carboxyl groups of glutamate by the platinate derivative. Briefly, the complex was formed by the following procedure. First, the active compound DDAP was weighed out and dissolved in 5% dextrose. After the DDAP solubilization step, aG6 was weighed out and added to
Figure BDA0002715283060000961
(solution)The solution was allowed to stir at 45-55 ℃ for 1 hour. The pH of the solution was adjusted to 6.5-7.0 using 1N NaOH and the solution was stirred for 1-2 hours. The formation of the complex was visually confirmed. However, when the pH was adjusted to an acidic pH of 3-5, the color returned to the original color, indicating decomplexing of the polyglutamated antifolate and cisplatin. Figure 15 depicts a schematic providing a possible scheme explaining the observed pH-dependent complex formation between polyglutamated antifolate and cisplatin.
Complex formation was confirmed using HPLC, which showed two distinct peaks that were combined into 1 large peak at high pH of 6.5 to 7.5 and then reappeared at low pH of 3-5. Repeated experiments in the absence of Captisol showed that complex formation was independent of
Figure BDA0002715283060000962
Production of pentaglutamated pemetrexed-DDAP complex (PGPD) liposomes
Briefly, PGPD was encapsulated in liposomes by the following procedure. First, the lipid components of the liposome membrane were weighed out and combined as a concentrated ethanol solution at a temperature of about 65 ℃. In this example, the lipids used were hydrogenated soy phosphatidylcholine, cholesterol and DSPE-PEG-2000(1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [ methoxy (polyethylene glycol) -2000 ]). The molar ratio of HSPC to cholesterol to PEG-DSPE is about 3:2: 0.15. Next, PGPD was prepared as described above. The PGPD drug solution was heated to 65 ℃. The ethanol lipid solution was injected into the PGPD solution using a small bore needle. During this step, the drug solution was thoroughly stirred using a magnetic stirrer. Mixing is performed at elevated temperatures (63-72 ℃) to ensure that the lipids are in a liquid crystalline state (as opposed to a gel state achieved at temperatures below the lipid transition temperature Tm-51-54 ℃). As a result, the lipids hydrate and form multiple bilayer (multilamellar) vesicles (MLVs) containing PGPD in the aqueous core.
Shrinking MLV using filter extrusion
MLVs are fragmented into monolayer (single bilayer) vesicles of the desired size by high pressure extrusion using two passes through a stack of (track etched polycarbonate) membranes. The stacked membrane had two layers with a pore size of 200nm and six layers with a pore size of 100 nm. During extrusion, the temperature is kept above Tm to ensure plasticity of the lipid film. Due to extrusion, the large, non-uniform MLV in size and layer becomes a small, uniform (100-120nm) unilamellar vesicle (ULV), which sequesters the drug inside. Hydrodynamic dimensions (diameter) at 25 ℃ were measured in a quartz microcuvette using a Malvern Zetasizer Nano ZS instrument (Southborough, MA) with a backscatter detector (90 °). Samples were diluted 50-fold in the formulation matrix prior to analysis.
And (3) purifying the liposome:
after generating PGPD-containing ULVs, the liposome-external PGPD is removed using tangential flow diafiltration against a suitable buffer for small volumes of column or for large volumes. Although many different buffers known in the art can be used, in this example the buffer used is 5mM HEPES, 145mM sodium chloride (pH 6.7). After completion of purification, filter sterilization was performed using a 0.22 micron filter. Liposomes prepared according to the above procedure were determined to have a diameter of 116.6nm, a PDI of 0.083 and a zeta potential of-2.05 mV.
Example 3: targeted liposomal polyglutamated pemetrexed cell delivery
Method of producing a composite material
Production of targeted gamma-hexaglutaminated pemetrexed (HGP) liposomes
Gamma HGP (gG6) was encapsulated in liposomes and the liposomes were reduced and purified according to the procedure essentially described in example 1.
Antibody conjugation
Activated liposomes were prepared by adding DSPE-PEG-maleimide to the lipid composition. Liposomes contain four different lipids: hydrogenated Soy Phosphatidylcholine (HSPC), cholesterol, 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [ methoxy (polyethylene glycol) -2000] (DSPE-PEG-2000) and 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [ maleimide (polyethylene glycol) -2000] (DSPE-PEG-maleimide) in a ratio of 3:2:0.1125: 0.0375.
Antibody thiolation is accomplished by linking a sulfhydryl group to a primary amine using Traut's reagent (2-iminothiolane). The antibody was suspended in PBS at a concentration of 0.9-1.6 mg/ml. Traut reagent (14mM) was added to the antibody solution at a final concentration of 1-5mM, and then removed by dialysis after incubation at room temperature for 1 hour. Thiolated antibody was added to the activated liposomes at a ratio of 60g/mol phospholipid, and the reaction mixture was incubated at room temperature for 1 hour and overnight with 4uL cysteine to terminate the reaction, and unconjugated antibody was removed by dialysis.
Exemplary direct and post-insertion antibody-liposome conjugation methods are provided below.
Exemplary antibody conjugation method 1: direct conjugation
The antibody or fragment thereof (e.g., Fab or scFv) can be conjugated directly to the thiol-reactive liposome. Thiol-reactive liposomes were prepared by adding DSPE-PEG-maleimide to the lipid composition. Liposomes contain four different lipids: hydrogenated Soy Phosphatidylcholine (HSPC), cholesterol, 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [ methoxy (polyethylene glycol) -2000] (DSPE-PEG-2000) and 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [ maleimide (polyethylene glycol) -2000] (DSPE-PEG-maleimide) in a ratio of 3:2:0.1125: 0.0375.
Thiolation of an antibody (or fragment thereof, such as a Fab or scFv) is accomplished by linking a sulfhydryl group to a primary amine using Traut's reagent (2-iminothiolane). The antibody (or fragment thereof) was suspended in PBS at a concentration of 0.9-1.6 mg/ml. Traut reagent (14mM) was added to the antibody (or fragment thereof) solution at a final concentration of 1-5mM, and then removed by dialysis after incubation at room temperature for 1 hour. The thiolated antibody (or fragment thereof) is added to the thiol-reactive liposomes at a ratio of 60g/mol phospholipid, and the reaction mixture is incubated at room temperature for 1 hour and at 4 ℃ overnight. L-cysteine was used to stop the reaction and unconjugated antibody (or fragment thereof) was removed by dialysis.
Antibodies or fragments thereof containing a cysteine residue at the C-terminus (such as Fab or scFv) can be conjugated directly to liposomes by incubating the reduced antibody (or fragment thereof) with thiol-reactive liposomes. Antibodies (or fragments thereof) with a cysteine tail are solubilized and reduced by 10-20mM reducing agents (such as 2-mercaptoethylamine, cysteine or dithioerythritol) at pH < 7. Excess reducing agent is removed completely by size exclusion chromatography or dialysis. The purified and reduced antibody (or fragment thereof) may be directly conjugated to a thiol-reactive liposome.
Exemplary antibody conjugation method 2: and (3) post insertion:
antibodies or fragments thereof containing a cysteine residue at the C-terminus (e.g., Fab or scFv) can be conjugated and incorporated into liposomes by "post-insertion" methods. Micelles of thiol-reactive lipopolymers (such as DSPE-PEG-maleimide) are prepared by dissolving in an aqueous solution at 10 mg/ml. Antibodies (or fragments thereof) with a cysteine tail are solubilized and reduced by 10-20mM reducing agents (such as 2-mercaptoethylamine, cysteine or dithioerythritol) at pH < 7. Excess reducing agent is removed completely by size exclusion chromatography or dialysis. The purified and reduced antibody (or fragment thereof) is then incubated with the micelles of thiol-reactive lipid polymer at a molar ratio of 1: 4. At the end of the reaction, the excess maleimide groups were quenched by a small amount of cysteine (1mM) or mercaptoethanol. Unconjugated antibody (or fragments thereof) is removed by size exclusion chromatography. The purified conjugated micelles are then incubated with liposomes at 37 ℃ or elevated temperature.
Physical characteristics of the nanoparticles
Figure BDA0002715283060000971
Figure BDA0002715283060000981
Dose response studies of HGP (pentaglutaminated pemetrexed) and liposomes.
Passage on day 3 (48 hours) and day 4 (72 hours)
Figure BDA0002715283060000982
(CTG) luminescence cell viability assay to determine cell viability. This assay determines the number of viable cells in culture based on quantifying the ATP present therein, which in turn indicates the presence of metabolically active cells. CTG assays use luciferase as readout. To assess cell viability, use was made of
Figure BDA0002715283060000983
Luminocyte viability assay study dose-response inhibition of pemetrexed, HGP and liposomes on growth of different cancer cells. Human cancer cells were harvested, counted and seeded at the same cell density on day 0. On day 1, a series of 8 dilutions of each test article was added to the cells. Dose response curves were generated and fitted using GraphPad Prism and the IC50 for each test article was calculated. The lower the IC50, the more effective the test article is in inhibiting cancer cell growth.
On day 0, cells were plated at 5x 10 per well4The cell density of each cell was seeded into 100. mu.l of fresh medium in a 96-well plate. 8 serial 2-fold dilutions of each test article in culture medium were generated and added to the cells in triplicate on day 1. In addition, cells from three wells were treated with vehicle only (HBS for free drug or empty liposomes for liposomal HGP) as control.
On day 3 and 4, 100. mu.l of each well was added
Figure BDA0002715283060000984
Reagents and incubated for 15 minutes at room temperature. Luciferase luminescence was recorded for each well. In addition, 8 serial 2-fold dilutions of vehicle (HBS or empty liposomes) in culture medium were added to the empty wells and included in the assay to generate background luminescence signal. Luciferase signals were normalized by subtracting the background luminescence signal from the readings, respectively.
Human normal primary bone marrow CD34+ cells were obtained from ATCC (ATCC accession No. PCS-800-012). Cells were thawed at 37 ℃ for 1 minute and then placed on ice. The cells were then resuspended in StemBan SFEM(Stem cell technology catalog number 9650) plus 10% heat-inactivated fetal bovine serum (Corning 35-015-CV). Cells were plated at 2.5 × 104The density of individual cells/well was seeded into 96-well culture plates. The following day, viable cells were collected via centrifugation and washed at 2.5x104The density of individual cells/well was resuspended in neutrophil growth medium (StemBan SFEM plus 10% heat-inactivated fetal bovine serum plus 100ng/ml human stem cell factor (Sigma Cat. No. H8416), 20ng/ml human granulocyte colony stimulating factor (Sigma Cat. No. H5541), and 10ng/ml human recombinant IL3(Sigma SRP 3090). cells were incubated at 37 ℃ for 10 days.fresh medium was added every two days 4The density of individual cells/well was seeded in 96-well plates and incubated overnight at 37 ℃. The following day, the test article or vehicle was resuspended in neutrophil growth medium and added to the plate. The cells were then incubated at 37 ℃ for 48 hours or 72 hours, and then assayed at each time point using the Cell Titer Glo assay (Promega catalog No. G7572).
The methods used for the cell lines AML12 (non-cancerous hepatocytes) and CCD841 (non-cancerous colonic epithelial cells) are similar to those used for cancer cells.
Results
Dose response relationships of free pemetrexed gamma hexaglutamate (gG6), (non-targeted) liposomal gamma hexaglutamate (liposomal gG6), pemetrexed, and folate receptor alpha-targeting antibody (FR1Ab) liposomal pemetrexed gamma hexaglutamate (liposomal gG6-FR1Ab) in the NCI H2342 non-small cell lung cancer (NSCLC) adenocarcinoma subtype are shown in FIG. 3. The output is the percentage of viable cells as measured by luciferase luminescence after 48 hours of treatment. As shown in fig. 3, free pemetrexed gG6 appears to be least potent as measured by IC 50. Both liposomal pemetrexed gG6 and liposomal pemetrexed gG6-FR1Ab were 7-fold and 40-fold more potent than free pemetrexed, respectively.
Similar data for HT-29 colon cancer cell lines are shown in figure 4, which depicts cell viability in percent. As shown in this figure, free pemetrexed gG6 appears to be least potent. In this case, the liposomal pemetrexed gG6 was twice as potent as pemetrexed, and the liposomal pemetrexed gG6-FR1Ab was 5 times more potent than free pemetrexed.
Example 4: in vivo studies
The method comprises the following steps:
safety studies in mice
Since some of the major toxicities associated with pemetrexed-based therapies are of the hematological and hepatic, it is important to evaluate the effect of liposome α G6(Lp-aG6) in an in vivo (murine) model and compare changes in the blood and hepatic serochemical groups after treatment. To obtain this data, an initial dose range study was performed using healthy female BALB/c mice (6-8 weeks old) purchased from The Jackson Laboratory (Bar Harbor, ME). Prior to the study, animals were weighed, randomly assigned by body weight, observed for clinical abnormalities, and divided into groups (5 mice per group). Doses from 10mg/kg up to 200mg/kg were studied to identify tolerable doses in mice. Treatment was administered intravenously once a week for four weeks. Body weight and detailed clinical observations were recorded daily. At the end of the study, mice were euthanized at day 28 and blood and tissue were harvested from untreated control mice as well as mice treated with 40mg/kg liposomal aG6 and 80mg/kg liposomal aG 6. Whole blood was collected into K2-EDTA anticoagulation tubes for comprehensive complete blood cell count (CBC), and serum was separated for comprehensive chemical analysis and sent to IDEXX (Westbrook, ME) on the day of collection.
Results：
In general, treatment with liposomal aG6 at two dose levels of 40mg/kg and 80mg/kg once per week for 4 weeks was well tolerated and there was no significant difference in body weight compared to untreated controls. To assess some of the effects on hematological parameters, White Blood Cell (WBC) counts, neutrophil counts, and platelet counts were measured after 4 weeks of treatment with liposomal aG6 at two dose levels of 40mg/kg and 80mg/kg administered once a week. As can be seen in fig. 16, there was no significant decrease in mean neutrophil, mean leukocyte and mean platelet counts after four weeks of treatment with liposome aG6 in treated animals compared to untreated control animals. Hemoglobin and reticulocyte indices were measured to assess the effect on erythrocytes. As shown in fig. 17, at the higher dose level, the mean hemoglobin concentration decreased minimally. In parallel, the mean reticulocyte proliferation index increased slightly, indicating the bone marrow response to treatment by increasing red blood cell production. Overall, this effect appeared to be minor, as the hemoglobin level of the mice was maintained after 4 weeks of treatment. These data taken together indicate that at these dosage levels of 40mg/kg and 80mg/kg once a week, there is little effect on bone marrow and related hematological indicators.
Another problem with pemetrexed is that hepatotoxicity is observed in some patients treated with pemetrexed-based therapies. To assess liver health in mice, serum chemistry including serum aspartate Aminotransferase (AST) and serum alanine Aminotransferase (ALT) and serum albumin was measured. As shown in figure 18, mean AST and mean ALT levels of hepatic transaminase did not increase significantly at 4 weeks compared to untreated controls after 4 weeks of treatment with liposome aG6 at both dose levels of 40mg/kg and 80mg/kg administered once a week. The mean albumin level was also unchanged. These data taken together indicate an advantageous safety profile of liposome aG 6.
Preliminary experimental efficacy studies in mouse xenografts
To assess whether there was any tumor control after treatment with liposomal alpha pemetrexed G6(Lp-aG6), a preliminary study was conducted. In this study, immunodeficient female nude mice (Nu/J; 6-8 weeks old) were purchased from The Jackson Laboratory (Bar Harbor, ME). NCI-H292 (non-small cell lung cancer) cells were incubated at 37 ℃ with 5% CO2The incubators were cultured in RPMI medium supplemented with 10% fetal bovine serum. 1X 106One cell was inoculated subcutaneously into the flank of the back of each mouse. Tumor volume and body weight were monitored twice weekly. Tumor-bearing mice were randomly assigned by tumor volume on day 0 and divided into groups (5 mice per group): control, pemetrexed and liposome aG 6. Pemetrexed was administered intravenously every three weeks at a dose of 167 mg/kg. This murine dose of 167mg/kg every three weeks is equivalent to every three FDA/EMA approved 500mg/m per week2Human dosage and schedule. Liposomal aG6 was administered intravenously at 80mg/kg once a week for 4 weeks. Tumor size was measured with calipers and tumor burden was calculated using the following equation: tumor volume is 0.5x (tumor length) x (tumor width)2(ii) a Relative tumor volume (tumor volume/tumor volume at day 0) x 100%. This study was still in progress, but preliminary data are shown in fig. 19, in this figure, the relative tumor volumes after treatment with liposome aG6 and pemetrexed are shown. As can be seen from these preliminary data, the liposome aG6 provided better tumor control than pemetrexed.
Other embodiments are as follows:
in a non-limiting embodiment of the present disclosure, a composition is provided comprising alpha polyglutamated methotrexate.
In the composition of the previous paragraph, the composition may comprise pentaglutamated or hexaglutamated methotrexate.
In the composition of any of the preceding two paragraphs, the composition may comprise alpha polyglutamated methotrexate, which may include pentaglutamated or hexaglutamated methotrexate.
Non-limiting exemplary liposomal alpha polyglutamated methotrexate (L-alpha PMTX) compositions can include compositions of any of the first three segments, and the liposomes can optionally be pegylated (PL-alpha PMTX).
In the L- α PMTX or PL- α PMTX compositions of the previous paragraph, the α polyglutamated methotrexate may comprise pentaglutamated or hexaglutamated methotrexate.
In the L-alpha PMTX or PL-alpha PMTX compositions of either of the preceding two paragraphs, the liposomes can be anionic or neutral.
In the L- α PMTX or PL- α PMTX compositions of any of the preceding three paragraphs, a targeting moiety can be attached to one or both of PEG and the exterior of the liposome, and the targeting moiety can have specific affinity for a surface antigen on a target cell of interest (TL- α PMTX or TPL- α PMTX).
In the L- α PMTX or PL- α PMTX compositions of any of the first four paragraphs, the targeting moiety can be attached to one or both of PEG and the exterior of the liposome and can be a polypeptide.
In the L- α PMTX or PL- α PMTX composition of any of the first five paragraphs, the targeting moiety may be attached to one or both of PEG and the exterior of the liposome, and may be an antibody or a fragment of an antibody.
In the L- α PMTX or PL- α PMTX composition of any of the first six paragraphs, one or more of an immunostimulatory agent, a detectable label, and a maleimide may be disposed on at least one of the PEG and the exterior of the liposome.
In the L-alpha PMTX or PL-alpha PMTX composition of any one of the first seven paragraphs, e.g. using
Figure BDA0002715283060001001
The polypeptide may be 0.5x 10 as determined by the assay-10To 10x 10-6Equilibrium dissociation constants (Kd) within the range bind to the antigen.
In the L- α PMTX or PL- α PMTX composition of any of the preceding eight paragraphs, the polypeptide can specifically bind to one or more folate receptors selected from the group consisting of: folate receptor alpha (FR-. alpha.), folate receptor beta (FR-. beta.), and folate receptor (FR-).
A non-limiting exemplary method of killing a hyperproliferative cell, comprising contacting the hyperproliferative cell with the liposomal α polyglutamated methotrexate composition of any one of the first nine paragraphs.
In the method of the previous paragraph, the hyperproliferative cell is a cancer cell.
A non-limiting exemplary method for treating cancer comprises administering to a subject having or at risk of having cancer an effective amount of an alpha polyglutamated methotrexate composition of any one of the preceding paragraphs of the eleventh through third preceding paragraphs.
In the method of the previous paragraph, the cancer may be one or more selected from the group consisting of: lung cancer, pancreatic cancer, breast cancer, ovarian cancer, lung cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colon cancer, esophageal cancer, cervical cancer, renal cancer, bile duct cancer, gallbladder cancer, and hematologic malignancies.
A non-limiting exemplary maintenance therapy for a subject undergoing or having undergone cancer therapy comprises administering to the subject undergoing or having undergone cancer therapy an effective amount of the alpha polyglutamated methotrexate composition of any one of the preceding paragraphs of the first thirteenth through fifth paragraphs.
A non-limiting exemplary pharmaceutical composition can comprise any of the alpha polyglutamated methotrexate compositions of part IV.
A non-limiting exemplary method for treating an immune system disorder can comprise administering to a subject having or at risk of having an immune system disorder an effective amount of an alpha-polyglutamated methotrexate composition of any one of the preceding paragraphs of the fourteenth to sixth paragraphs above.
A non-limiting exemplary method for treating an infection can include administering to a subject having or at risk of having an infectious disease an effective amount of an alpha-polyglutamated methotrexate composition of any one of the preceding paragraphs of the first fifteenth paragraph to the first seventh paragraph.
A non-limiting exemplary method of delivering alpha-polyglutamated methotrexate to a tumor that expresses a folate receptor on its surface can comprise administering to a subject having the tumor an amount of the polyglutamated methotrexate composition of any one of the preceding paragraphs of the first sixteenth through first eighth paragraphs to deliver a therapeutically effective dose of the alpha-polyglutamated methotrexate to the tumor.
A non-limiting exemplary method of making a liposomal α -polyglutamated methotrexate composition (the liposomal α -polyglutamated methotrexate composition comprising an α -polyglutamated methotrexate composition of any of the preceding paragraphs of the first seventeenth paragraph through the first ninth paragraph) includes forming a mixture comprising a liposomal composition, α -polyglutamated methotrexate, in a solution; homogenizing the mixture to form liposomes in the solution; and processing the mixture to form liposomes containing the polyglutamated methotrexate.
A non-limiting exemplary pharmaceutical composition comprises an alpha-polyglutamated methotrexate composition of any one of the preceding paragraphs of the first eighteenth paragraph through the first tenth paragraph.
While the disclosure has been described with reference to various embodiments, it will be understood that various modifications may be made without departing from the spirit of the disclosure. The scope of the disclosure should, therefore, be determined with reference to the appended claims, along with the full scope of equivalents to which such claims are entitled. Throughout this application, various publications are referred to by author name and date or by patent number or patent publication number. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled in the art as of the date of the invention described and claimed herein. However, citation of a reference herein shall not be construed as an admission that such reference is prior art to the present invention.
Various novel chemical entities, methods and apparatus for preparing these chemical entities are set forth in the following appended claims.
It should be understood that the detailed description section, and not the summary and abstract sections, is intended to be used to interpret the claims. The summary and abstract sections may set forth one or more, but not all exemplary embodiments of the invention as contemplated by the inventors, and are, therefore, not intended to limit the invention and the appended claims in any way.
Us application No. 62/627,703 filed on 7.2.2018; us application No. 62/627,714 filed on 7.2.2018; us application No. 62/627,716 filed on 7.2.2018; us application No. 62/627,731 filed on 7.2.2018; us application No. 62/627,741 filed on 7.2.2018; us application No. 62/630,629 filed on 14/2/2018; us application No. 62/630,634 filed on 14/2/2018; us application No. 62/630,637 filed on 14/2/2018; us application No. 62/630,671 filed on 14/2/2018; us application No. 62/630,713 filed on 14/2/2018; us application No. 62/630,728 filed on 14/2/2018; us application No. 62/630,744 filed on 14/2/2018; us application No. 62/630,820 filed on 14/2/2018; us application No. 62/630,825 filed on 14/2/2018; us application No. 62/636,294 filed on 28.2.2018; us application No. 62/662,374 filed on 25.4.2018; us application No. 62/702,732 filed 24.7.2018; us application No. 62/702,561 filed 24.7.2018; us application No. 62/764,943 filed on 8/17 in 2018; and us application No. 62/764,955 filed 2018, 8, 17, the disclosures of each are hereby incorporated by reference in their entirety.
Sequence listing
<110> L.E.A.F. HOLDINGS GROUP company (L.E.A.F. HOLDING GROUP LLC)
C.Nixsa (NIYIKIZA, Clet)
V.M. Moxiabout (MOYO, Victor M.)
<120> alpha polyglutamated methotrexate and use thereof
<130> 6155.0089
<140> to be distributed
<141> accompanying submission
<150> US 62/627,716
<151> 2018-02-07
<150> US 62/627,741
<151> 2018-02-07
<150> US 62/630,728
<151> 2018-02-14
<150> US 62/662,374
<151> 2018-04-25
<150> US 62/702,732
<151> 2018-07-24
<150> US 62/764,943
<151> 2018-08-17
<160> 44
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<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> synthetic construct
<400> 22
Leu Gly Ile Pro Glu Ala Glu Tyr
1 5
<210> 23
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> synthetic construct
<400> 23
Leu Gly Ile Pro Glu Gln Ala Tyr
1 5
<210> 24
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> synthetic construct
<400> 24
Leu Gly Ile Ala Glu Ala Glu Tyr
1 5
<210> 25
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> synthetic construct
<400> 25
Leu Gly Ile Pro Glu Ala Ala Tyr
1 5
<210> 26
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> synthetic construct
<400> 26
Leu Gly Ile Ala Glu Gln Ala Tyr
1 5
<210> 27
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> synthetic construct
<400> 27
Leu Gly Ile Ala Glu Ala Ala Tyr
1 5
<210> 28
<211> 18
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> synthetic construct
<400> 28
Leu Leu Ile Ile Leu Arg Arg Arg Ile Arg Lys Gln Ala His Ala His
1 5 10 15
Ser Lys
<210> 29
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> synthetic construct
<400> 29
Leu Lys Ala Leu Ala Ala Leu Ala Lys Lys Ile Leu
1 5 10
<210> 30
<211> 18
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> synthetic construct
<400> 30
Lys Leu Ala Leu Lys Leu Ala Leu Lys Ala Leu Lys Ala Ala Leu Lys
1 5 10 15
Leu Ala
<210> 31
<211> 21
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> synthetic construct
<400> 31
Lys Glu Thr Trp Trp Glu Thr Trp Trp Thr Glu Trp Ser Gln Pro Lys
1 5 10 15
Lys Lys Arg Lys Val
20
<210> 32
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> synthetic construct
<400> 32
Asp His Gln Leu Asn Pro Ala Phe
1 5
<210> 33
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> synthetic construct
<400> 33
Asp Pro Lys Gly Asp Pro Lys Gly
1 5
<210> 34
<211> 18
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> synthetic construct
<400> 34
Val Thr Val Thr Val Thr Val Thr Val Thr Gly Lys Gly Asp Pro Lys
1 5 10 15
Pro Asp
<210> 35
<211> 16
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> synthetic construct
<400> 35
Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys
1 5 10 15
<210> 36
<211> 13
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> synthetic construct
<400> 36
Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro Pro Gln
1 5 10
<210> 37
<211> 27
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> synthetic construct
<400> 37
Gly Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu Gly Lys Ile Asn Leu
1 5 10 15
Lys Ala Leu Ala Ala Leu Ala Lys Lys Ile Leu
20 25
<210> 38
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> synthetic construct
<400> 38
Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg
1 5 10
<210> 39
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> synthetic construct
<400> 39
Arg Arg Arg Arg Arg Arg Arg
1 5
<210> 40
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> synthetic construct
<400> 40
Arg Arg Arg Arg Arg Arg Arg Arg
1 5
<210> 41
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> synthetic construct
<400> 41
Arg Arg Arg Arg Arg Arg Arg Arg Arg
1 5
<210> 42
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> synthetic construct
<400> 42
Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg
1 5 10
<210> 43
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> synthetic construct
<400> 43
Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg
1 5 10
<210> 44
<211> 33
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> synthetic construct
<220>
<221> MISC _ feature
<222> (33)..(33)
<223> Xaa can be 2 to 15 Arg independently in L and/or D form
<400> 44
Tyr Thr Ile Trp Met Pro Glu Asn Pro Arg Pro Gly Thr Pro Cys Asp
1 5 10 15
Ile Phe Thr Asn Ser Arg Gly Lys Arg Ala Ser Asn Gly Gly Gly Gly
20 25 30
Xaa

Claims (94)

1. A composition comprising alpha polyglutamated methotrexate, wherein at least one glutamyl group has an alpha carboxyl linkage.
2. The composition of claim 1, wherein the alpha polyglutamated methotrexate comprises 1-10 glutamyl groups having an alpha carboxy linkage.
3. The composition of claim 1 or 2, wherein the alpha polyglutamated methotrexate contains 4, 5, 2-10, 4-6, or more than 5 glutamyl groups.
4. The composition according to any one of claims 1 to 3, comprising alpha tetraglutamated methotrexate.
5. The composition of any one of claims 1-3, comprising alpha pentaglutamated methotrexate.
6. The composition of any one of claims 1 to 3, comprising alpha-hexaglutaminated methotrexate.
7. The composition according to any one of claims 1 to 6, wherein
(a) Two or more glutamyl groups have an alpha carboxyl linkage,
(b) each glutamyl group, other than the glutamyl group of methotrexate, has an alpha carboxyl linkage; or
(c) Two or more glutamyl groups have a gamma carboxyl linkage,
8. the composition of any one of claims 1 to 7, wherein at least one glutamyl group has both an alpha carboxyl linkage and a gamma carboxyl linkage.
9. The composition according to any one of claims 1 to 8, wherein
(a) At least 2 glutamyl groups of the alpha polyglutamated methotrexate are in the L form,
(b) each glutamyl group of the alpha polyglutamated methotrexate is in the L form,
(c) at least 1 glutamyl group of said alpha polyglutamated methotrexate is in the D form,
(d) each glutamyl group of said alpha polyglutamated methotrexate is in the D form, in addition to the glutamyl group of methotrexate, or
(e) At least 2 glutamyl groups of the alpha polyglutamated methotrexate are in the L form, and at least 1 glutamyl group is in the D form.
10. The composition according to any one of claims 1 to 9, wherein the polyglutamate is linear.
11. The composition according to any one of claims 1 to 9, wherein the polyglutamate is branched.
12. A liposome composition comprising alpha polyglutamated methotrexate (Lp-alpha PMTX) according to any one of claims 1 to 11.
13. The L α PP composition of claim 12, wherein the α polyglutamated methotrexate comprises glutamyl in the L form having an α carboxyl linkage.
14. The Lp-alpha PMTX composition of claim 12 or 13, wherein each glutamyl group of the alpha polyglutamated methotrexate is in the L form.
15. The Lp-alpha PMTX composition of claim 12 or 13, wherein at least one glutamyl group of the alpha polyglutamated methotrexate is in the D form.
16. The Lp-alpha PMTX composition of any of claims 12-15, wherein the liposomes comprise alpha polyglutamated methotrexate containing 4, 5, 2-10, 4-6, or more than 5 glutamyl groups.
17. The Lp-alpha PMTX composition of any of claims 12-16, wherein at least one glutamyl group of the alpha polyglutamated methotrexate has a gamma carboxyl linkage.
18. The composition of any one of claims 12-17, wherein at least one glutamyl group has both an alpha carboxyl linkage and a gamma carboxyl linkage.
19. The composition of any one of claims 12-18, which contains 2, 3, 4, 5, 2-10, 4-6, or more than 5 glutamyl groups having both alpha and gamma carboxyl linkages.
20. The Lp-alpha PMTX composition of any of claims 12-19, wherein the liposomes comprise alpha polyglutamated methotrexate containing alpha tetraglutamated methotrexate, alpha pentaglutamated methotrexate, or alpha hexaglutamated methotrexate.
21. The Lp-alpha PMTX composition of any of claims 12-19, wherein the liposomes comprise alpha polyglutamated methotrexate containing alpha tetraglutamated methotrexate, alpha pentaglutamated methotrexate, or alpha hexaglutamated methotrexate.
22. The Lp-alpha PMTX composition of any of claims 12-21, wherein the polyglutamate is linear or branched.
23. The Lp-alpha PMTX composition of any one of claims 12-22, wherein the liposome is pegylated (pa Lp-alpha PMTX).
24. The Lp-alpha PMTX composition of any of claims 12-23, wherein the liposomes comprise at least 1% weight/weight (w/w) of the alpha polyglutamated methotrexate, or wherein at least 1% of the starting material of alpha polyglutamated MTX is encapsulated (embedded) in the alpha PMTX during the process of preparing the Lp-alpha PMTX.
25. The Lp-alpha PMTX composition of any one of claims 12-24, wherein the diameter of the liposomes is in the range of 20nm to 500nm or 20nm to 200 nm.
26. The Lp-alpha PMTX composition of any of claims 12-25, wherein the diameter of the liposomes is in the range of 80nm to 120 nm.
27. The Lp-alpha PMTX composition of any one of claims 12-26, wherein the liposomes are formed from liposomal components.
28. The Lp-alpha PMTX composition of 27, wherein the liposomal composition comprises at least one of anionic lipids and neutral lipids.
29. The Lp-alpha PMTX composition of 27 or 28, wherein the liposome component comprises at least one selected from the group consisting of: DSPE; DSPE-PEG; DSPE-PEG-maleimide; HSPC; HSPC-PEG; cholesterol; cholesterol-PEG; and cholesterol-maleimide.
30. The Lp-alpha PMTX composition of any of claims 27-29, wherein the liposome component comprises at least one selected from the group consisting of: DSPE; DSPE-PEG; DSPE-PEG-FITC; DSPE-PEG-maleimide; cholesterol; and HSPC.
31. The Lp-alpha PMTX composition of any one of claims 27-30, wherein one or more liposomal components further comprise a steric stabilizer.
32. The Lp-alpha PMTX composition of 31, wherein the steric stabilizer is at least one selected from the group consisting of: polyethylene glycol (PEG); poly-L-lysine (PLL); monosialoganglioside (GM 1); poly (vinyl pyrrolidone) (PVP); poly (acrylamide) (PAA); poly (2-methyl-2-oxazoline); poly (2-ethyl-2-oxazoline); a phosphatidylpolyglycerol; poly [ N- (2-hydroxypropyl) methacrylamide ]; amphiphilic poly-N-vinylpyrrolidone; an L-amino acid-based polymer; oligomerization of glycerol; copolymers comprising polyethylene glycol and polypropylene oxide; poloxamer 188; and polyvinyl alcohol.
33. The Lp- α PMTX composition of claim 32, wherein the steric stabilizer is PEG, and the PEG has a number average molecular weight (Mn) of 200 to 5000 daltons.
34. The Lp-alpha PMTX composition of any of claims 12-33, wherein the liposome is anionic or neutral.
35. The Lp-alpha PMTX composition of any of claims 12-33, wherein the liposome has a zeta potential less than or equal to zero.
36. The Lp-alpha PMTX composition of any of claims 12-33, wherein the liposomes have a zeta potential between 0 to-150 mV.
37. The Lp-alpha PMTX composition of any of claims 12-33, wherein the liposomes have a zeta potential between-30 to-50 mV.
38. The Lp-alpha PMTX composition of any of claims 12-33, wherein the liposome is cationic.
39. The Lp-alpha PMTX composition of any one of claims 12-38, wherein the liposome has an interior space comprising the alpha polyglutamated methotrexate and a pharmaceutically acceptable aqueous carrier.
40. The Lp- α PMTX composition of 39, wherein the pharmaceutically acceptable carrier comprises a tonicity agent, such as dextrose, mannitol, glycerol, potassium chloride, sodium chloride, at a concentration of greater than 1%.
41. The Lp- α PMTX composition of 39, wherein the pharmaceutically acceptable aqueous carrier is trehalose.
42. The Lp- α PMTX composition of claim 41, wherein the pharmaceutically acceptable carrier comprises 1% to 50% trehalose.
43. The Lp-alpha PMTX composition of any one of claims 39-42, wherein the pharmaceutically acceptable carrier comprises a 1% to 50% dextrose solution.
44. The Lp-alpha PMTX composition of any one of claims 39-43, wherein the interior space of the liposome comprises 5% dextrose suspended in HEPES buffer solution.
45. The Lp-alpha PMTX composition of any one of claims 39-44, wherein the pharmaceutically acceptable carrier comprises a buffer, such as HEPES Buffered Saline (HBS) or the like, at a concentration of between 1 and 200mM and at a pH of between 2 and 8.
46. The Lp-alpha PMTX composition of any one of claims 39-45, wherein the pharmaceutically acceptable carrier comprises sodium acetate and calcium acetate in a total concentration between 50mM to 500 mM.
47. The Lp-alpha PMTX composition of any of claims 12-46, wherein the interior space of the liposomes has a pH of 5-8 or a pH of 6-7, or any range therebetween.
48. The Lp-alpha PMTX composition of any one of claims 12-47, wherein the liposomes comprise less than 500,000 or less than 200,000 of the alpha polyglutamated methotrexate molecules.
49. The Lp-alpha PMTX composition of any one of claims 12-48, wherein the liposomes comprise between 10 to 100,000 of the alpha polyglutamated methotrexate molecules, or any range therebetween.
50. The Lp-a PMTX composition of any one of claims 12-49, further comprising a targeting moiety, and wherein the targeting moiety has specific affinity for a surface antigen on a target cell of interest.
51. The Lp-a PMTX composition of claim 50, wherein the targeting moiety is attached to one or both of PEG and the exterior of the liposome, optionally wherein targeting moiety is attached to one or both of the PEG and the exterior of the liposome by a covalent bond.
52. The Lp-alpha PMTX composition of claim 50 or 51, wherein the targeting moiety is a polypeptide.
53. The Lp-alpha PMTX composition of any one of claims 50-52, wherein the targeting moiety is an antibody or an antigen-binding fragment of an antibody.
54. The Lp-alpha PMTX composition of any one of claims 50-53, wherein, e.g., using
Figure FDA0002715283050000031
The targeting moiety was determined by assay to be at 0.5x10-10To 10x10-6An equilibrium dissociation constant (Kd) in a range binds the surface antigen.
55. The Lp-alpha PMTX composition of any one of claims 50-55, wherein the targeting moiety specifically binds to one or more folate receptors selected from the group consisting of: folate receptor alpha (FR-. alpha.), folate receptor beta (FR-. beta.), and folate receptor (FR-).
56. The Lp-alpha PMTX composition of any one of claims 50-56, wherein the targeting moiety comprises one or more selected from the group consisting of: antibodies, humanized antibodies, antigen-binding fragments of antibodies, single chain antibodies, single domain antibodies, bispecific antibodies, synthetic antibodies, pegylated antibodies, and multimeric antibodies.
57. The Lp-alpha PMTX composition of any one of claims 50-56, wherein each pegylated liposome comprises 1 to 1000 or 30 to 200 targeting moieties.
58. The Lp-a PMTX composition of any one of claims 39-57, further comprising one or more of an immunostimulatory agent, a detectable label, and a maleimide, wherein the immunostimulatory agent, the detectable label, or the maleimide is attached to the exterior of the PEG or the liposome.
59. The Lp-alpha PMTX composition of claim 58, wherein the immunostimulatory agent is at least one selected from the group consisting of: a protein immunostimulant; a nucleic acid immunostimulant; a chemical immunostimulant; a hapten; and an adjuvant.
60. The Lp-alpha PMTX composition of claim 58 or 59, wherein the immunostimulatory agent is at least one selected from the group consisting of: fluorescein; fluorescein Isothiocyanate (FITC); DNP; beta glucan; beta-1, 3-glucan; beta-1, 6-glucan; resolvins (e.g., resolvins D such as D)n-6DPAOr Dn-3DPAResolvin E, or T series resolvin); and Toll-like receptor (TLR) modulators, such as oxidized low density lipoproteins (e.g., OXPAC, PGPC) and eritoran lipids (e.g., E5564).
61. The Lp-alpha PMTX composition of any one of claims 58-60, wherein the immunostimulatory agent and the detectable label are the same.
62. The Lp-alpha PMTX composition of any one of claims 58-61, further comprising a hapten.
63. The Lp-alpha PMTX composition of claim 62, wherein the hapten comprises one or more of fluorescein or beta 1, 6-glucan.
64. The Lp-alpha PMTX composition of any of claims 12-63, further comprising at least one cryoprotectant selected from the group consisting of: mannitol; trehalose; sorbitol; and sucrose.
65. A targeted composition comprising the composition of any one of claims 1-64.
66. A non-targeted composition comprising the composition of any one of claims 1-49.
67. The Lp-a PMTX composition of any one of claims 12-66, further comprising carboplatin and/or pembrolizumab.
68. A pharmaceutical composition comprising the liposomal alpha-polyglutamated methotrexate composition of any one of claims 12-67.
69. A pharmaceutical composition comprising the alpha polyglutamated methotrexate composition of any one of claims 1-7.
70. The composition of any one of claims 1-69, for use in the treatment of a disease.
71. Use of the composition of any one of claims 1-70 in the manufacture of a medicament for treating a disease.
72. A method for treating or preventing a disease in a subject in need of such treatment or prevention, the method comprising administering to the subject the composition of any one of claims 1-70.
73. A method for treating or preventing a disease in a subject in need of such treatment or prevention, the method comprising administering to the subject the liposomal alpha polyglutamated methotrexate composition of any one of claims 12-69.
74. A method of killing a hyperproliferative cell, comprising contacting a hyperproliferative cell with the composition of any one of claims 1-69.
75. A method of killing a hyperproliferative cell, comprising contacting the hyperproliferative cell with the liposomal α -polyglutamated methotrexate composition of any one of claims 12-69.
76. The method of claim 74 or 75, wherein the hyperproliferative cell is a cancer cell, a mammalian cell, and/or a human cell.
77. A method for treating cancer, the method comprising administering to a subject having or at risk of having cancer an effective amount of the composition of any one of claims 1-69.
78. A method for treating cancer, the method comprising administering to a subject having or at risk of having cancer an effective amount of the liposomal alpha polyglutamated methotrexate composition of any one of claims 12-68.
79. The method of claim 77 or 78, wherein the cancer is selected from the group consisting of: non-hematologic malignancies including, for example, lung cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer, bile duct cancer, gall bladder cancer, sarcomas (e.g., osteosarcoma), brain cancer, central nervous system cancer, and melanoma; and hematological malignancies such as leukemia, lymphoma and other B cell malignancies, myeloma and other plasma cell dyscrasias.
80. The method of claim 77 or 78, wherein the cancer is a member selected from the group consisting of: optionally wherein the cancer. In some embodiments, the cancer is selected from the group consisting of: breast cancer, head and neck cancer, lung cancer, gastric cancer, osteosarcoma, non-hodgkin lymphoma (NHL), Acute Lymphoblastic Leukemia (ALL), mycosis fungoides (cutaneous T-cell lymphoma), choriocarcinoma and chorioadenoma, non-leukemic meningeal cancer, soft tissue sarcoma (desmoid, invasive fibromatosis), bladder cancer, and Central Nervous System (CNS) lymphoma.
81. The method of claim 77 or 78, wherein the cancer is mesothelioma or non-small cell lung cancer (NSCLC).
82. The method of claim 77 or 78, wherein the cancer is a sarcoma, such as osteosarcoma.
83. A method for treating cancer, the method comprising administering to a subject having or at risk of having a cancer cell that expresses on its surface a folate receptor bound by a targeting moiety, an effective amount of the Lp-alpha PMTX composition of any one of claims 50-66.
84. A maintenance therapy for a subject undergoing or having undergone cancer therapy, the maintenance therapy comprising administering to a subject undergoing or having undergone cancer therapy an effective amount of the composition of any one of claims 1-69.
85. A maintenance therapy for a subject undergoing or having undergone cancer therapy, the maintenance therapy comprising administering to the subject undergoing or having undergone cancer therapy an effective amount of the liposomal α -polyglutamated methotrexate composition of any one of claims 12-69.
86. A method for treating an immune system disorder, the method comprising administering to a subject having or at risk of having an immune system disorder an effective amount of the composition of any one of claims 1-69.
87. A method for treating an immune system disorder, the method comprising administering to a subject having or at risk of having an immune system disorder an effective amount of the liposomal α -polyglutamated methotrexate composition of any one of claims 8-69.
88. A method for treating the following diseases:
(a) an infectious disease, the method comprising administering to a subject having or at risk of having an infectious disease an effective amount of the composition of any one of claims 1-69;
(b) an infectious disease, a cardiovascular disease, a metabolic disease, or another disease, the method comprising administering to a subject having or at risk of having an infectious disease, a cardiovascular disease, or another disease an effective amount of the composition of any one of claims 1-59, wherein the disease is a member selected from: atherosclerosis, cardiovascular disease (CVD), coronary artery disease, myocardial infarction, stroke, metabolic syndrome, gestational trophoblastic disease, and ectopic pregnancy;
(c) An autoimmune disease, the method comprising administering to a subject having or at risk of having an autoimmune disease an effective amount of the composition of any one of claims 1-59;
(d) rheumatoid arthritis, the method comprising administering to a subject having or at risk of having rheumatoid arthritis an effective amount of the composition of any one of claims 1-59;
(e) an inflammatory disorder, the method comprising administering to a subject having or at risk of having inflammation an effective amount of a composition of any one of claims 1-59, optionally wherein the inflammation is acute, chronic and/or systemic inflammation; or
(f) A skin condition, the method comprising administering to a subject having or at risk of having a skin condition an effective amount of the composition of any one of claims 1-59, optionally wherein the skin condition is psoriasis.
89. A method for treating an infectious disease, the method comprising administering to a subject having or at risk of having an infectious disease an effective amount of the liposomal alpha polyglutamated methotrexate composition of any one of claims 12-69.
90. A method of delivering alpha polyglutamated methotrexate to a tumor that expresses a folate receptor on the surface, the method comprising: administering to a subject having the tumor an amount of the Lp-alpha PMTX composition of any one of claims 1-69 to deliver a therapeutically effective dose of the alpha polyglutamated methotrexate to the tumor.
91. A method of making an alpha polyglutamated methotrexate composition comprising the liposomal alpha polyglutamated methotrexate composition of any one of claims 12-69, the method comprising: forming a mixture comprising a liposome component and an alpha polyglutamated antifolate agent in solution; homogenizing the mixture to form liposomes in the solution; and processing the mixture to form liposomes containing alpha-polyglutamated methotrexate.
92. A method of making the composition of any one of claims 12-69, the method comprising the steps of: forming a mixture comprising a liposome component and alpha-polyglutamated methotrexate in a solution; homogenizing the mixture to form liposomes in the solution; processing the mixture to form liposomes encapsulating and/or encapsulating alpha polyglutamated methotrexate; and providing a targeting moiety on a surface of the liposome, the targeting moiety having a specific affinity for at least one of folate receptor alpha (FR-alpha), folate receptor beta (FR-beta), and folate receptor (FR-).
93. The method of claim 92, wherein the processing step comprises one or more of: film hydration, extrusion, on-line mixing, ethanol injection technology, freeze-thaw technology, reverse phase evaporation, dynamic high-pressure micro-jet, micro-jet mixing, multiple emulsion method, freeze-drying multiple emulsion method, 3D printing, membrane contactor method and stirring.
94. The method of claim 92, wherein the processing step comprises one or more steps of altering the size of the liposomes by one or more of extrusion, high pressure microfluidization, and/or sonication steps.
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